PYK2 related polynucleotide products

ABSTRACT

The present invention features a method for treatment of an organism having a disease or condition characterized by an abnormality in a signal transduction pathway, wherein the signal transduction pathway includes a PYK2 protein. The invention also features methods for diagnosing such diseases and for screening for agents that will be useful in treating such diseases, The invention also feature purified and/or isolated nucleic acid encoding a PYK2 protein.

FIELD OF THE INVENTION

The present invention relates generally to the fields of biology,biochemistry and medicine and more specifically to the field of cellularsignal transduction.

BACKGROUND OF THE INVENTION

None of the following discussion of the background of the invention isadmitted to be prior art to the invention.

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of tyrosineresidues on proteins. The phosphorylation state of a protein is modifiedthrough the reciprocal actions of tyrosine phosphatases (TPs) andtyrosine kinases (TKs), including receptor tyrosine kinases andnon-receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) belong to a family of transmembraneproteins and have been implicated in cellular signaling pathways. Thepredominant biological activity of some RTKs is the stimulation of cellgrowth and proliferation, while other RTKs are involved in arrestinggrowth and promoting differentiation. In some instances, a singletyrosine kinase can inhibit, or stimulate, cell proliferation dependingon the cellular environment in which it is expressed.

RTKs are composed of at least three domains: an extracellular ligandbinding domain, a transmembrane domain and a cytoplasmic catalyticdomain that can phosphorylate tyrosine residues. Ligand binding tomembrane-bound receptors induces the formation of receptor dimers andallosteric changes that activate the intracellular kinase domains andresult in the self-phosphorylation (autophosphorylation and/ortransphosphorylation) of the receptor on tyrosine residues. Individualphosphotyrosine residues of the cytoplasmic domains of receptors mayserve as specific binding sites that interact with a host of cytoplasmicsignaling molecules, thereby activating various signal transductionpathways.

The intracellular, cytoplasmic, non-receptor protein tyrosine kinases donot contain a hydrophobic transmembrane domain or an extracellulardomain and share non-catalytic domains in addition to sharing theircatalytic kinase domains. Such non-catalytic domains include the SH2domains and SH3 domains. The non-catalytic domains are thought to beimportant in the regulation of protein-protein interactions duringsignal transduction.

A central feature of signal transduction is the reversiblephosphorylation of certain proteins. Receptor phosphorylation stimulatesa physical association of the activated receptor with target molecules,which either are or are not phosphorylated.

Some of the target molecules such as phospholipase Cγ are in turnphosphorylated and activated. Such phosphorylation transmits a signal tothe cytoplasm. Other target molecules are not phosphorylated, but assistin signal transmission by acting as adapter molecules for secondarysignal transducer proteins. For example, receptor phosphorylation andthe subsequent allosteric changes in the receptor recruit the Grb-2/SOScomplex to the catalytic domain of the receptor where its proximity tothe membrane allows it to activate ras.

The secondary signal transducer molecules generated by activatedreceptors result in a signal cascade that regulates cell functions suchas cell division or differentiation. Reviews describing intracellularsignal transduction include Aaronson, Science, 254:1146-1153, 1991;Schlessinger, Trends Biochem. Sci., 13:443-47, 1988; and Ullrich andSchlessinger, Cell, 61:203-212, 1990.

Signal transduction pathways that regulate ion channels (e.g., potassiumchannels and calcium channels) involve G proteins which function asintermediaries between receptors and effectors. Gilman, Ann. Rev.Biochem., 56:615-649 (1987); Brown and Birnbaumer, Ann. Rev. Physiol.,52:197-213 (1990). G-coupled protein receptors are receptors forneurotransmitters, ligands that are responsible for signal production innerve cells as well as for regulation of proliferation anddifferentiation of nerves and other cell types. Neurotransmitterreceptors exist as different subtypes which are differentially expressedin various tissues and neurotransmitters such as acetylcholine evokeresponses throughout the central and peripheral nervous systems.

The muscarinic acetylcholine receptors play important roles in a varietyof complex neural activities such as learning, memory, arousal and motorand sensory modulation. These receptors have also been implicated inseveral central nervous system disorders such as Alzheimer's disease,Parkinson's disease, depression and schizophrenia.

Some agents that are involved in a signal transduction pathwayregulating one ion channel, for example a potassium channel, may also beinvolved in one or more other pathways regulating one or more other ionchannels, for example a calcium channel. Dolphin, Ann. Rev. Physiol.,52:243-55 (1990); Wilk-Blaszczak et al., Neuron, 12:109-116 (1994). Ionchannels may be regulated either with or without a cytosolic secondmessenger. Hille, Neuron, 9:187-195 (1992). One possible cytosolicsecond messenger is a tyrosine kinase. Huang et al., Cell, 75:1145-1156(1993), incorporated herein by reference in its entirety, including anydrawings.

The receptors involved in the signal transduction pathways that regulateion channels are ultimately linked to the ion channels by variousintermediate events and agents. For example, such events include anincrease in intracellular calcium and inositol triphosphate andproduction of endothelin. Frucht, et al., Cancer Research, 52:1114-1122(1992); Schrey, et al., Cancer Research, 52:1786-1790 (1992).Intermediary agents include bombesin, which stimulates DNA synthesis andthe phosphorylation of a specific protein kinase C substrate.Rodriguez-Pena, et al., Biochemical and Biophysical ResearchCommunication, 140(1):379-385 (1986); Fisher and Schonbrunn, The Journalof Biological Chemistry, 263(6):2208-2816 (1988).

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinaselocalized to focal adhesions that is known to associate with two Srcfamily kinases. Schaller, et al., Proc. Natl. Acad. Sci. U.S.A.,89:5192-5196 (1992), incorporated herein by reference in its entirety,including any drawings; Cobb et al., Molecular and Cellular Biology,14(1):147-155 (1994). The proteins associated with the cytoplasmicsurface of adhesion molecules are reviewed in Gumbiner, Neuron,11:551-564 (1993).

FAK may regulate interactions of integrins, agonist receptors, and/orstress fibers. Shattil et al., The Journal of Biological Chemistry,269(20):14738-14745 (1994); Ridley and Hall, The EMBO Journal,13(11):2600-2610 (1994). FAK does not contain SH2 or SH3 domains and theamino acid sequence of FAK is highly conserved among birds, rodents andman.

In some cells the C-terminal domain of FAK is expressed autonomously asa 41 kDa protein called FRNK and the 140 C-terminal residues of FAKcontain a focal adhesion targeting (FAT) domain. The cDNA's encodingFRNK are given in Schaller et al., Molecular and Cellular Biology,13(2):785-791 (1993), incorporated herein by reference in its entirety,including any drawings. The FAT domain was identified and said to berequired for localization of FAK to cellular focal adhesions inHilderbrand et al., The Journal of Cell Biology, 123(4):993-1005 (1993).

SUMMARY OF THE INVENTION

The present invention relates to PYK2 polypeptides, nucleic acidsencoding such polypeptides, cells, tissues and animals containing suchpolypeptides and nucleic acids, antibodies to such polypeptides, assaysutilizing such polypeptides, and methods relating to all of theforegoing. PYK2 polypeptides are involved in various signal transductionpathways and thus the present invention provides several agents andmethods useful for diagnosing, treating, and preventing various diseasesor conditions associated with abnormalities in these pathways.

The present invention is based upon the identification and isolation ofa novel non-receptor tyrosine kinase, termed PYK2, that is activated bybinding of ligand to G-coupled protein receptors such as bradykinin andacetylcholine. PYK2 has a predicted molecular weight of 111 kD andcontains five domains: (1) a relatively long N-terminal domain; (2) akinase catalytic domain; (3) a proline rich domain; (4) another prolinerich domain; and (5) a C-terminal focal adhesion targeting (FAT) domain.PYK2 does not contain a SH2 or SH3 domain.

The FAT domain of PYK2 has 62% similarity to the FAT domain of anothernon-receptor tyrosine kinase, FAK, which is also activated by G-coupledproteins. The overall similarity between PYK2 and FAK is 52%. PYK2 isexpressed principally in neural tissues, although expression can also bedetected in hematopoietic cells at early stages of development and insome tumor cell lines. The expression of PYK2 does not correspond withthe expression of FAK.

PYK2 is believed to regulate the activity of potassium channels inresponse to neurotransmitter signalling. PYK2 enzymatic activity ispositively regulated by phosphorylation on tyrosine and results inresponse to binding of bradykinin, TPA, calcium ionophore, carbachol,TPA+forskolin, and membrane depolarization. The combination of toxinsknown to positively regulate G-coupled receptor signalling (such aspertusis toxin, cholera toxins, TPA and bradykinin) increases thephosphorylation of PYK2.

Activated PYK2 phosphorylates RAK, a delayed rectifier type potassiumchannel, and thus suppresses RAK activity. In the same system, FAK doesnot phosphorylate RAK. PYK2 is responsible for regulatingneurotransmitter signalling and thus may be used to treat conditions ofnervous system by enhancing or inhibiting such signalling.

Thus, in a first aspect the invention features an isolated, purified,enriched or recombinant nucleic acid encoding a PYK2 polypeptide.

By "isolated" in reference to nucleic acid is meant a polymer of 2(preferably 21, more preferably 39, most preferably 75) or morenucleotides conjugated to each other, including DNA or RNA that isisolated from a natural source or that is synthesized. The isolatednucleic acid of the present invention is unique in the sense that it isnot found in a pure or separated state in nature. Use of the term"isolated" indicates that a naturally occurring sequence has beenremoved from its normal cellular environment. Thus, the sequence may bein a cell-free solution or placed in a different cellular environment.The term does not imply that the sequence is the only nucleotide chainpresent, but does indicate that it is the predominate sequence present(at least 10-20% more than any other nucleotide sequence) and isessentially free (about 90-95% pure at least) of non-nucleotide materialnaturally associated with it. Therefore, the term does not encompass anisolated chromosome encoding a PYK2 polypeptide.

By the use of the term "enriched" in reference to nucleic acid is meantthat the specific DNA or RNA sequence constitutes a significantly higherfraction (2-5 fold) of the total DNA or RNA present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat enriched does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased in a useful manner andpreferably separate from a sequence library. The term significant hereis used to indicate that the level of increase is useful to the personmaking such an increase, and generally means an increase relative toother nucleic acids of about at least 2 fold, more preferably at least 5to 10 fold or even more. The term also does not imply that there is noDNA or RNA from other sources. The other source DNA may, for example,comprise DNA from a yeast or bacterial genome, or a cloning vector suchas pUC19. This term distinguishes from naturally occurring events, suchas viral infection, or tumor type growths, in which the level of onemRNA may be naturally increased relative to other species of mRNA. Thatis, the term is meant to cover only those situations in which a personhas intervened to elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term "purified" in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/ml). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones could be obtained directly from total DNA or from totalRNA. The cDNA clones are not naturally occurring, but rather arepreferably obtained via manipulation of a partially purified naturallyoccurring substance (messenger RNA). The construction of a cDNA libraryfrom mRNA involves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁶ -foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated.

By "a PYK2 polypeptide" is meant two or more contiguous amino acids setforth in the full length amino acid sequence of SEQ ID NO:1. The PYK2polypeptide can be encoded by a full-length nucleic acid sequence or anyportion of the full-length nucleic acid sequence, so long as afunctional activity of the polypeptide is retained. Preferred functionalactivities include the ability to phosphorylate and regulate RAK and/orother potassium channels.

In preferred embodiments the isolated nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence set forth in thefull length nucleic acid sequence SEQ ID NO:2 or at least 27, 30, 35, 40or 50 contiguous nucleotides thereof and the PYK2 polypeptide comprises,consists essentially of, or consists of at least 9, 10, 15, 20, or 30contiguous amino acids of a PYK2 polypeptide.

By "comprising" it is meant including, but not limited to, whateverfollows the word "comprising". Thus, use of the term "comprising"indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. By"consisting of" is meant including, and limited to, whatever follows thephrase "consisting of". Thus, the phrase "consisting of" indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By "consisting essentially of" is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase "consisting essentially of" indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

Compositions and probes of the present invention may contain humannucleic acid encoding a PYK-2 polypeptide but are substantially free ofnucleic acid not encoding a human PYK-2 polypeptide. The human nucleicacid encoding a PYK-2 polypeptide is at least 18 contiguous bases of thenucleotide sequence set forth in SEQ ID NO 2 and will selectivelyhybridize to human genomic DNA encoding a PYK-2 polypeptide, or iscomplementary to such a sequence. The nucleic acid may be isolated froma natural source by cDNA cloning or subtractive hybridization; thenatural source may be blood, semen, and tissue of various organismsincluding eukaryotes, mammals, birds, fish, plants, gorillas, rhesusmonkeys, chimpanzees and humans; and the nucleic acid may be synthesizedby the triester method or by using an automated DNA synthesizer. In yetother preferred embodiments the nucleic acid is a conserved or uniqueregion, for example those useful for the design of hybridization probesto facilitate identification and cloning of additional polypeptides, thedesign of PCR probes to facilitate cloning of additional polypeptides,and obtaining antibodies to polypeptide regions.

By "conserved nucleic acid regions", are meant regions present on two ormore nucleic acids encoding a PYK2 polypeptide, to which a particularnucleic acid sequence can hybridize to under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening for nucleic acid encoding PYK2 polypeptides are provided inAbe, et al. J. Biol. Chem., 19:13361 (1992) (hereby incorporated byreference herein in its entirety, including any drawings). Preferably,conserved regions differ by no more than 7 out of 20 nucleotides.

By "unique nucleic acid region" is meant a sequence present in a fulllength nucleic acid coding for a PYK2 polypeptide that is not present ina sequence coding for any other naturally occurring polypeptide. Suchregions preferably comprise 12 or 20 contiguous nucleotides present inthe full length nucleic acid encoding a PYK2 polypeptide.

The invention also features a nucleic acid probe for the detection of aPYK2 polypeptide or nucleic acid encoding a PYK2 polypeptide in asample. The nucleic acid probe contains nucleic acid that will hybridizeto a sequence set forth in SEQ ID NO:2.

In preferred embodiments the nucleic acid probe hybridizes to nucleicacid encoding at least 12, 27, 30, 35, 40 or 50 contiguous amino acidsof the full-length sequence set forth in SEQ ID NO:1. Various low orhigh stringency hybridization conditions may be used depending upon thespecificity and selectivity desired.

By "high stringency hybridization conditions" is meant those hybridizingconditions that (1) employ low ionic strength and high temperature forwashing, for example, 0.015M NaCl/0.0015M sodium citrate/0.1% SDS at 50°C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(3) employ 50% formamide, 5×SSC (0.75M NaCl, 0.075M Sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS. Under stringent hybridization conditionsonly highly complementary nucleic acid sequences hybridize. Preferably,such conditions prevent hybridization of nucleic acids having 1 or 2mismatches out of 20 contiguous nucleotides.

Methods for using the probes include detecting the presence or amountPYK2 RNA in a sample by contacting the sample with a nucleic acid probeunder conditions such that hybridization occurs and detecting thepresence or amount of the probe bound to PYK2 RNA. The nucleic acidduplex formed between the probe and a nucleic acid sequence coding for aPYK2 polypeptide may be used in the identification of the sequence ofthe nucleic acid detected (for example see, Nelson et al., inNonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego(Kricka, ed., 1992) hereby incorporated by reference herein in itsentirety, including any drawings). Kits for performing such methods maybe constructed to include a container means having disposed therein anucleic acid probe.

The invention also features recombinant nucleic acid, preferably in acell or an organism. The recombinant nucleic acid may contain a sequenceset forth in SEQ ID NO:2 and a vector or a promoter effective toinitiate transcription in a host cell. The recombinant nucleic acid canalternatively contain a transcriptional initiation region functional ina cell, a sequence complimentary to an RNA sequence encoding a PYK2polypeptide and a transcriptional termination region functional in acell.

In another aspect the invention features an isolated, enriched orpurified PYK2 polypeptide.

By "isolated" in reference to a polypeptide is meant a polymer of 2(preferably 7, more preferably 13, most preferably 25) or more aminoacids conjugated to each other, including polypeptides that are isolatedfrom a natural source or that are synthesized. The isolated polypeptidesof the present invention are unique in the sense that they are not foundin a pure or separated state in nature. Use of the term "isolated"indicates that a naturally occurring sequence has been removed from itsnormal cellular environment. Thus, the sequence may be in a cell-freesolution or placed in a different cellular environment. The term doesnot imply that the sequence is the only amino acid chain present, butthat it is the predominate sequence present (at least 10-20% more thanany other sequence) and is essentially free (about 90-95% pure at least)of non-amino acid material naturally associated with it.

By the use of the term "enriched" in reference to a polypeptide is meantthat the specific amino acid sequence constitutes a significantly higherfraction (2-5 fold) of the total of amino acids present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other amino acids present, or bya preferential increase in the amount of the specific amino acidsequence of interest, or by a combination of the two. However, it shouldbe noted that enriched does not imply that there are no other amino acidsequences present, just that the relative amount of the sequence ofinterest has been significantly increased. The term significant here isused to indicate that the level of increase is useful to the personmaking such an increase, and generally means an increase relative toother amino acids of about at least 2 fold, more preferably at least 5to 10 fold or even more. The term also does not imply that there is noamino acid from other sources. The other source amino acid may, forexample, comprise amino acid encoded by a yeast or bacterial genome, ora cloning vector such as pUC19.

The term is meant to cover only those situations in which man hasintervened to elevate the proportion of the desired amino acid.

It is also advantageous for some purposes that an amino acid sequence bein purified form. The term "purified" in reference to a polypeptide doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural levelthis level should be at least 2-5 fold greater, e.g., in terms ofmg/ml). Purification of at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. The substance is preferably free ofcontamination at a functionally significant level, for example 90%, 95%,or 99% pure.

In preferred embodiments the PYK-2 polypeptide contains at least 9, 10,15, 20, or 30 contiguous amino acids of the full-length sequence setforth in SEQ ID NO:1.

In yet another aspect the invention features a purified antibody (e.g.,a monoclonal or polyclonal antibody) having specific binding affinity toa PYK2 polypeptide. The antibody contains a sequence of amino acids thatis able to specifically bind to a PYK2 polypeptide.

By "specific binding affinity" is meant that the antibody will bind to aPYK-2 polypeptide at a certain detectable amount but will not bind otherpolypeptides, such as FAK polypeptides to the same extent, underidentical conditions.

Antibodies having specific binding affinity to a PYK2 polypeptide may beused in methods for detecting the presence and/or amount of a PYK2polypeptide in a sample by contacting the sample with the antibody underconditions such that an immunocomplex forms and detecting the presenceand/or amount of the antibody conjugated to the PYK2 polypeptide.Diagnostic kits for performing such methods may be constructed toinclude a first container means containing the antibody and a secondcontainer means having a conjugate of a binding partner of the antibodyand a label.

In another aspect the invention features a hybridoma which produces anantibody having specific binding affinity to a PYK2 polypeptide.

By "hybridoma" is meant an immortalized cell line which is capable ofsecreting an antibody, for example a PYK2 antibody.

In preferred embodiments the PYK2 antibody comprises a sequence of aminoacids that is able to specifically bind a PYK2 polypeptide.

Another aspect of the invention features a method of detecting thepresence or amount of a compound capable of binding to a PYK2polypeptide. The method involves incubating the compound with a PYK2polypeptide and detecting the presence or amount of the compound boundto the PYK2 polypeptide.

In preferred embodiments, the compound inhibits a phosphorylationactivity of PYK2 and is selected from the group consisting oftyrphostins, quinazolines, quinaxolines, and quinolines. The presentinvention also features compounds capable of binding and inhibiting PYK2polypeptide that are identified by methods described above.

In another aspect the invention features a method of screening potentialagents useful for treatment of a disease or condition characterized byan abnormality in a signal transduction pathway that contains aninteraction between a PYK2 polypeptide and a natural binding partner(NBP). The method involves assaying potential agents for those able topromote or disrupt the interaction as an indication of a useful agent.

By "screening" is meant investigating an organism for the presence orabsence of a property. The process may include measuring or detectingvarious properties, including the level of signal transduction and thelevel of interaction between a PYK2 polypeptide and a NBP.

By "disease or condition" is meant a state in an organism, e.g., ahuman, which is recognized as abnormal by members of the medicalcommunity. The disease or condition may be characterized by anabnormality in one or more signal transduction pathways in a cell,preferably a cell listed in table 1, wherein one of the components ofthe signal transduction pathway is either a PYK2 polypeptide or a NBP.

                  TABLE 1                                                         ______________________________________                                                              LEVEL                                                               CHECKED   OF EX-                                                  CELL LINE   BY        PRESSION  COMMENT                                       ______________________________________                                        NIH3T3      IP/IB     -         High expression of                                                            Fak (IP/IB)                                   L cells     IP/IB     -         --                                            Jurkat      IP/IB     +         very high expression                          (T-lymphoblastic cells)         of Fak (IP/IB)                                KG-1        IP/IB     +++       --                                            (human myeloblast/                                                            promyelocte)                                                                  K562 (human IP/IB     ++        --                                            erythroleukemia)                                                              CHRF        IP/IB     +++       After differentiation                         (premegakaryocyte,              with TPA for 3                                human)                          days, mobility shift                                                          of PYK2                                       L8057       IP/IB     ++        After differentiation                         (premegakaryocyte,              with TPA for 3                                mouse)                          days, mobility shift                                                          of PYK2                                       T47D (human breast                                                                        IP/IB, PCR                                                                              ++        PCR gave higher                               carcinoma)                      expression as                                                                 compared to the IP/                                                           IB                                            GH3 (Pituitary tumor,                                                                     IP/IB     ++        --                                            rat)                                                                          PC12        IP/IB, PCR,                                                                             +++       No change of PYK2                                         Northern            expression level or                                                           mobility after 36 hr                                                          treatment with NGF,                                                           No expression of                                                              Fak (IP/IB)                                   XC (Sarcoma, rat)                                                                         IP/IB     +++       PYK2 is                                                                       phosphorylated on                                                             tyrosine                                      HEL (human  IP/IB     ++        --                                            erythroleukemia                                                               /myeloid)                                                                     HL-60 (human                                                                              IP/IB     ++++      --                                            promyelocytic                                                                 leukemia)                                                                     NG108-15    IP/IB     +         --                                            (neuroblastoma-                                                               glioma hybrid)                                                                ______________________________________                                    

Specific diseases or disorders which might be treated or prevented,based upon the affected cells include: myasthenia gravis; neuroblastoma;disorders caused by neuronal toxins such as cholera toxin, pertusistoxin, or snake venom; acute megakaryocytic myelosis; thrombocytopenia;those of the central nervous system such as seizures, stroke, headtrauma, spinal cord injury, hypoxia-induced nerve cell damage such as incardiac arrest or neonatal distress, epilepsy, neurodegenerativediseases such as Alzheimer's disease, Huntington's disease andParkinson's disease, dementia, muscle tension, depression, anxiety,panic disorder, obsessive-compulsive disorder, post-traumatic stressdisorder, schizophrenia, neuroleptic malignant syndrome, and Tourette'ssyndrome. Conditions that may be treated by PYK2 inhibitors includeepilepsy, schizophrenia, extreme hyperactivity in children, chronicpain, and acute pain. Examples of conditions that may be treated by PYK2enhancers (for example a phosphatase inhibitor) include stroke,Alzheimer's, Parkinson's, other neurodegenerative diseases and migraine.

Preferred disorders include epilepsy, stroke, schizophrenia, andParkinson's disorder as there is an established relationship betweenthese disorders and the function of potassium channels. See, McLean etal., Epilepsia 35:S5-S9 1994; Ricard-Mousnier et al., NeurophysiologieClinique 23:395-421, 1993; Crit Rev. Veurobiol 7:187-203, 1994; Simonand Lin, Biophys. J. 64:A100, 1993; Birnstiel et al., Synapse (N.Y.)11:191-196, 1992; Coleman et al., Brain Res. 575:138-142 1992; Popolipet al., Br. J. Pharmacol 104:907-913, 1991; Murphy et al., Exp. BrainRes. 84:355-358, 1991; Rutecki et al., Epilepsia 32:1-2, 1991; Fisherand Coyle (ed), Frontiers of Clinical Neurosciene, Vol. 11"Neurotransmitters and Epilepsy"; Meeting, Woods Hole Mass., USAIX+260P. John Wiley and Sons, Inc. N.Y., N.Y.; Treherne and Ashford,Neuroscience 40:523-532, 1991; Gehlert, Prog. Neuro-Psychopharmacol.Biol. Psychiatry 18:1093-1102, 1994; Baudy, Expert Opin Ther. Pat. 19944/4:343-378; Porter and Rogawski, Epilepsia 33:S1-S6, 1992; Murphy, J.Physiol. 453:167-183, 1992; Cromakalim, Drugs Future 17/3:237-239, 1992;Carmeliet, Eur. Heart J. 12:30-37, 1991; Olpe et al., Experientia47/3:254-257, 1991; Andrade et al., Science 234/4781:1261-1265, 1986;Forster, J. Neurosci. Methods 13/3-4:199-212, 1985.

In preferred embodiments, the methods described herein involveidentifying a patient in need of treatment. Those skilled in the artwill recognize that various techniques may be used to identify suchpatients. For example, cellular potassium levels may be measured or theindividuals' genes may be examined for a defect.

By "abnormality" is meant a level which is statistically different fromthe level observed in organisms not suffering from such a disease orcondition and may be characterized as either an excess amount, intensityor duration of signal or a deficient amount, intensity or duration ofsignal. The abnormality in signal transduction may be realized as anabnormality in cell function, viability or differentiation state. Wehave determined that such abnormality in a pathway can be alleviated byaction at the PYK2:NBP interaction site in the pathway. An abnormalinteraction level may also either be greater or less than the normallevel and may impair the normal performance or function of the organism.Thus, it is also possible to screen for agents that will be useful fortreating a disease or condition, characterized by an abnormality in thesignal transduction pathway, by testing compounds for their ability toaffect the interaction between a PYK2 polypeptide and a NBP, since thecomplex formed by such interaction is part of the signal transductionpathway.

However, the disease or condition may be characterized by an abnormalityin the signal transduction pathway even if the level of interactionbetween the PYK2 polypeptide and NBP is normal.

By "interact" is meant any physical association between polypeptides,whether covalent or non-covalent. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. Examples ofnon-covalent bonds include electrostatic bonds, hydrogen bonds, and Vander Waals bonds. Furthermore, the interactions between polypeptides mayeither be direct or indirect. Thus, the association between two givenpolypeptides may be achieved with an intermediary agent, or several suchagents, that connects the two proteins of interest (e.g., a PYK2polypeptide and a NBP). Another example of an indirect interaction isthe independent production, stimulation, or inhibition of both a PYK2polypeptide and NBP by a regulatory agent. Depending upon the type ofinteraction present, various methods may be used to measure the level ofinteraction. For example, the strengths of covalent bonds are oftenmeasured in terms of the energy required to break a certain number ofbonds (i.e., kcal/mol) Non-covalent interactions are often described asabove, and also in terms of the distance between the interactingmolecules. Indirect interactions may be described in a number of ways,including the number of intermediary agents involved, or the degree ofcontrol exercised over the PYK2 polypeptide relative to the controlexercised over the NBP.

By "disrupt" is meant that the interaction between the PYK2 polypeptideand NBP is reduced either by preventing expression of the PYK2polypeptide, or by preventing expression of the NBP, or by specificallypreventing interaction of the naturally synthesized proteins or byinterfering with the interaction of the proteins.

By "promote" is meant that the interaction between a PYK2 polypeptideand NBP is increased either by increasing expression of a PYK2polypeptide, or by increasing expression of a NBP, or by decreasing thedephosphorylating activity of the corresponding regulatory TP (or otherphosphatase acting on other phosphorylated signalling components) bypromoting interaction of the PYK2 polypeptide and NBP or by prolongingthe duration of the interaction. Covalent binding can be promoted eitherby direct condensation of existing side chains or by the incorporationof external bridging molecules. Many bivalent or polyvalent linkingagents are useful in coupling polypeptides, such as an antibody, toother molecules. For example, representative coupling agents can includeorganic compounds such as thioesters, carbodiimides, succinimide esters,diisocyanates, glutaraldehydes, diazobenzenes and hexamethylenediamines. This listing is not intended to be exhaustive of the variousclasses of coupling agents known in the art but, rather, is exemplary ofthe more common coupling agents. (See Killen and Lindstrom 1984, J.Immunol. 133:1335-2549; Jansen, F. K., et al., 1982, Immunological Rev.62:185-15 216; and Vitetta et al., supra).

By "NBP" is meant a natural binding partner of a PYK2 polypeptide thatnaturally associates with a PYK2 polypeptide. The structure (primary,secondary, or tertiary) of the particular natural binding partner willinfluence the particular type of interaction between the PYK2polypeptide and the natural binding partner. For example, if the naturalbinding partner comprises a sequence of amino acids complementary to thePYK2 polypeptide, covalent bonding may be a possible interaction.Similarly, other structural characteristics may allow for othercorresponding interactions. The interaction is not limited to particularresidues and specifically may involve phosphotyrosine, phosphoserine, orphosphothreonine residues. A broad range of sequences may be capable ofinteracting with PYK2 polypeptides. Using techniques well known in theart, one may identify several natural binding partners for PYK2polypeptides.

By "signal transduction pathway" is meant the sequence of events thatinvolves the transmission of a message from an extracellular protein tothe cytoplasm through a cell membrane. The signal ultimately will causethe cell to perform a particular function, for example, touncontrollably proliferate and therefore cause cancer. Variousmechanisms for the signal transduction pathway (Fry et al., ProteinScience, 2:1785-1797, 1993) provide possible methods for measuring theamount or intensity of a given signal. Depending upon the particulardisease associated with the abnormality in a signal transductionpathway, various symptoms may be detected. Those skilled in the artrecognize those symptoms that are associated with the various otherdiseases described herein.

Furthermore, since some adapter molecules recruit secondary signaltransducer proteins towards the membrane, one measure of signaltransduction is the concentration and localization of various proteinsand complexes. In addition, conformational changes that are involved inthe transmission of a signal may be observed using circular dichroismand fluorescence studies.

In preferred embodiments the screening method involves growing cells(i.e., in a dish) that either naturally or recombinantly express aG-coupled protein receptor, PYK2, and RAK. The test compound is added ata concentration from 0.1 uM to 100 uM and the mixture is incubated from5 minutes to 2 hours. The ligand is added to the G-coupled proteinreceptor for preferably 5 to 30 minutes and the cells are lysed. RAK isisolated using immunoprecipitation or ELISA by binding to a specificmonoclonal antibody. The amount of phosphorylation compared to cellsthat were not exposed to a test compound is measured using ananti-phosphotyrosine antibody (preferably polyclonal). Examples ofcompounds that could be tested in such screening methods includetyrphostins, quinazolines, quinoxolines, and quinolines.

The quinazolines, tyrphostins, quinolines, and quinoxolines referred toabove include well known compounds such as those described in theliterature. For example, representative publications describingquinazoline include Barker et al., EPO Publication No. 0 520 722 A1;Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum andComer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO PublicationNo. 0 562 734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin etal., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178; Fry etal., Science 265:1093-1095 (1994); Jackman et al., Cancer Research51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee andSkibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem.54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975);Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Miniet al., Cancer Research 45:325-330 (1985); Phillips and Castle, J.Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., CancerResearch 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. andImmunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all ofwhich are incorporated herein by reference in their entirety, includingany drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

Quinolines are described in Dolle et al., J. Med. Chem. 37:2627-2629(1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al., J.Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem.Letters 2:1771-1774 (1992), all of which are incorporated by referencein their entirety, including any drawings.

Tyrphostins are described in Allen et al., Clin. Exa. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., "J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282(1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson etal., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683(1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro andThomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. andExperimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J.Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., CancerResearch 51:4430-4435 (1991); all of which are incorporated herein byreference in their entirety, including any drawings.

In another aspect the invention features a method of diagnosis of anorganism for a disease or condition characterized by an abnormality in asignal transduction pathway that contains an interaction between a PYK2polypeptide and a NBP. The method involves detecting the level ofinteraction as an indication of said disease or condition.

By "organism" is meant any living creature. The term includes mammals,and specifically humans. Preferred organisms include mice, as theability to treat or diagnose mice is often predictive of the ability tofunction in other organisms such as humans.

By "diagnosis" is meant any method of identifying a symptom normallyassociated with a given disease or condition. Thus, an initial diagnosismay be conclusively established as correct by the use of additionalconfirmatory evidence such as the presence of other symptoms. Currentclassification of various diseases and conditions is constantly changingas more is learned about the mechanisms causing the diseases orconditions. Thus, the detection of an important symptom, such as thedetection of an abnormal level of interaction between PYK2 polypeptidesand NBPs may form the basis to define and diagnose a newly named diseaseor condition. For example, conventional cancers are classified accordingto the presence of a particular set of symptoms. However, a subset ofthese symptoms may both be associated with an abnormality in aparticular signalling pathway, such as the ras²¹ pathway and in thefuture these diseases may be reclassified as ras²¹ pathway diseasesregardless of the particular symptoms observed.

Yet another aspect of the invention features a method for treatment ofan organism having a disease or condition characterized by anabnormality in a signal transduction pathway. The signal transductionpathway contains an interaction between a PYK2 polypeptide and a NBP andthe method involves promoting or disrupting the interaction, includingmethods that target the PYK2:NBP interaction directly, as well asmethods that target other points along the pathway.

In preferred embodiments the signal transduction pathway regulates anion channel, for example, a potassium ion, the disease or conditionwhich is diagnosed or treated are those described above, the agent is adominant negative mutant protein provided by gene therapy or otherequivalent methods as described below and the agent is therapeuticallyeffective and has an EC₅₀ or IC₅₀ as described below.

By "dominant negative mutant protein" is meant a mutant protein thatinterferes with the normal signal transduction pathway. The dominantnegative mutant protein contains the domain of interest (e.g., an PYK2polypeptide or a NBP), but has a mutation preventing proper signaling,for example by preventing binding of a second domain from the sameprotein. One example of a dominant negative protein is described inMillauer et al., Nature Feb. 10, 1994. The agent is preferably a peptidewhich blocks or promotes interaction of the PYK2 polypeptide and theNBP. The peptide may be recombinant, purified, or placed in apharmaceutically acceptable carrier or diluent.

An EC₅₀ or IC₅₀ of less than or equal to 100 μM is preferable, and evenmore preferably less than or equal to 50 μM, and most preferably lessthat or equal to 20 μM. Such lower EC₅₀ 's or IC₅₀ 's are advantageoussince they allow lower concentrations of molecules to be used in vivo orin vitro for therapy or diagnosis. The discovery of molecules with suchlow EC₅₀ 's and IC₅₀ 's enables the design and synthesis of additionalmolecules having similar potency and effectiveness. In addition, themolecule may have an EC₅₀ or IC₅₀ less than or equal to 100 μM at one ormore, but not all cells chosen from the group consisting of parathyroidcell, bone osteoclast, juxtaglomerular kidney cell, proximal tubulekidney cell, distal tubule kidney cell, cell of the thick ascending limbof Henle's loop and/or collecting duct, central nervous system cell,keratinocyte in the epidermis, parafollicular cell in the thyroid(C-cell), intestinal cell, trophoblast in the placenta, platelet,vascular smooth muscle cell, cardiac atrial cell, gastrin-secretingcell, glucagon-secreting cell, kidney mesangial cell, mammary cell, betacell, fat/adipose cell, immune cell and GI tract cell.

By "therapeutically effective amount" is meant an amount of apharmaceutical composition having a therapeutically relevant effect. Atherapeutically relevant effect relieves to some extent one or moresymptoms of the disease or condition in the patient; or returns tonormal either partially or completely one or more physiological orbiochemical parameters associated with or causative of the disease orcondition. Generally, a therapeutically effective amount is betweenabout 1 nmole and 1 μmole of the molecule, depending on its EC₅₀ or IC₅₀and on the age and size of the patient, and the disease associated withthe patient.

In other aspects, the invention provides transgenic, nonhuman mammalscontaining a transgene encoding a PYK2 polypeptide or a gene effectingthe expression of a PYK2 polypeptide. Such transgenic nonhuman mammalsare particularly useful as an in vivo test system for studying theeffects of introducing a PYK2 polypeptide, regulating the expression ofa PYK2 polypeptide (i.e., through the introduction of additional genes,antisense nucleic acids, or ribozymes).

A "transgenic animal" is an animal having cells that contain DNA whichhas been artificially inserted into a cell, which DNA becomes part ofthe genome of the animal which develops from that cell. Preferredtransgenic animals are primates, mice, rats, cows, pigs, horses, goats,sheep, dogs and cats. The transgenic DNA may encode for a human PYK2polypeptide. Native expression in an animal may be reduced by providingan amount of anti-sense RNA or DNA effective to reduce expression of thereceptor.

In another aspect, the invention describes a polypeptide comprising arecombinant PYK2 polypeptide or a unique fragment thereof. By "uniquefragment," is meant an amino acid sequence present in a full-length PYK2polypeptide that is not present in any other naturally occurringpolypeptide. Preferably, such a sequence comprises 6 contiguous aminoacids present in the full sequence. More preferably, such a sequencecomprises 12 contiguous amino acids present in the full sequence. Evenmore preferably, such a sequence comprises 18 contiguous amino acidspresent in the full sequence.

By "recombinant PYK2 polypeptide" is meant to include a polypeptideproduced by recombinant DNA techniques such that it is distinct from anaturally occurring polypeptide either in its location (e.g., present ina different cell or tissue than found in nature), purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature.

In another aspect, the invention describes a recombinant cell or tissuecontaining a purified nucleic acid coding for a PYK2 polypeptide. Insuch cells, the nucleic acid may be under the control of its genomicregulatory elements, or may be under the control of exogenous regulatoryelements including an exogenous promoter. By "exogenous" it is meant apromoter that is not normally coupled in vivo transcriptionally to thecoding sequence for the PYK2 polypeptide.

In another aspect, the invention features a PYK2 polypeptide bindingagent able to bind to a PYK2 polypeptide. The binding agent ispreferably a purified antibody which recognizes an epitope present on aPYK2 polypeptide. Other binding agents include molecules which bind tothe PYK2 polypeptide and analogous molecules which bind to a PYK2polypeptide.

By "purified" in reference to an antibody is meant that the antibody isdistinct from naturally occurring antibody, such as in a purified form.Preferably, the antibody is provided as a homogeneous preparation bystandard techniques. Uses of antibodies to the cloned polypeptideinclude those to be used as therapeutics, or as diagnostic tools.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 shows a schematic representation of the PYK2 domains (including akinase domain, a proline rich domain, and a Fat domain) and potentialbinding sites (including YAEI, YLNV, and YVWV).

FIG. 2 shows a possible mechanism for the membrane depolarization andcalcium influx that stimulate MEK and MAP kinase via activation of Ras.In PC12 cells, membrane depolarization leads to calcium influx throughL-type calcium channels and activates MAP kinase. Calcium influx leadsto activation of Ras and the activation of MAP in response to calciuminflux is inhibited by a dominant negative mutant of Ras.

Table 1 shows the expression pattern and levels of PYK2 in various celllines as checked by multiple methods.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to PYK2 polypeptides, nucleic acidsencoding such polypeptides, cells, tissues and animals containing suchnucleic acids, antibodies to such polypeptides, assays utilizing suchpolypeptides, and methods relating to all of the foregoing. Thoseskilled in the art will recognize that many of the methods describedbelow in relation to PYK-2, a NBP, or a complex of PYK-2 and a NBP couldalso be utilized with respect to the other members of this group.

I. Nucleic Acid Encoding A PYK2 Polypeptide.

Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons which specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan, the known aminoacids can be coded for by more than one codon. Thus, portions or all ofthe PYK2 gene could be synthesized to give a nucleic acid sequencesignificantly different from that shown in SEQ ID NO: 2. The encodedamino acid sequence thereof would, however, be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5'-end and/or the 3'-end of the nucleic acidformula shown in SEQ ID NO: 2 or a derivative thereof. Any nucleotide orpolynucleotide may be used in this regard, provided that its addition,deletion or substitution does not alter the amino acid sequence of SEQID NO:1 which is encoded by the nucleotide sequence. For example, thepresent invention is intended to include any nucleic acid sequenceresulting from the addition of ATG as an initiation codon at the 5'-endof the inventive nucleic acid sequence or its derivative, or from theaddition of TTA, TAG or TGA as a termination codon at the 3'-end of theinventive nucleotide sequence or its derivative. Moreover, the nucleicacid molecule of the present invention may, as necessary, haverestriction endonuclease recognition sites added to its 5'-end and/or3'-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequence of the PYK2 genes and fragmentsthereof permitted by the genetic code are, therefore, included in thisinvention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

II. A Nucleic Acid Probe for the Detection of PYK2.

A nucleic acid probe of the present invention may be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain another nucleic acid molecule of the present invention. Achromosomal DNA or cDNA library may be prepared from appropriate cellsaccording to recognized methods in the art (cf. Molecular Cloning: ALaboratory Manual, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989).

In the alternative, chemical synthesis is carried out in order to obtainnucleic acid probes having nucleotide sequences which correspond toN-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. Thus, the synthesized nucleic acid probes maybe used as primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, A Guide to Methods and Applications, edited by Michael etal., Academic Press, 1990, utilizing the appropriate chromosomal or cDNAlibrary to obtain the fragment of the present invention.

One skilled in the art can readily design such probes based on thesequence disclosed herein using methods of computer alignment andsequence analysis known in the art (cf. Molecular Cloning: A LaboratoryManual, second edition, edited by Sambrook, Fritsch, & Maniatis, ColdSpring Harbor Laboratory, 1989). The hybridization probes of the presentinvention can be labeled by standard labeling techniques such as with aradiolabel, enzyme label, fluorescent label, biotin-avidin label,chemiluminescence, and the like. After hybridization, the probes may bevisualized using known methods.

The nucleic acid probes of the present invention include RNA, as well asDNA probes, such probes being generated using techniques known in theart. The nucleic acid probe may be immobilized on a solid support.Examples of such solid supports include, but are not limited to,plastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, and acrylic resins, such as polyacrylamide and latexbeads. Techniques for coupling nucleic acid probes to such solidsupports are well known in the art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

III. Probe Based Method And Kit For Detecting PYK2.

One method of detecting the presence of PYK2 in a sample comprises a)contacting said sample with the above-described nucleic acid probe,under conditions such that hybridization occurs, and b) detecting thepresence of said probe bound to said nucleic acid molecule. One skilledin the art would select the nucleic acid probe according to techniquesknown in the art as described above. Samples to be tested include butshould not be limited to RNA samples of human tissue.

A kit for detecting the presence of PYK2 in a sample comprises at leastone container means having disposed therein the above-described nucleicacid probe. The kit may further comprise other containers comprising oneor more of the following: wash reagents and reagents capable ofdetecting the presence of bound nucleic acid probe. Examples ofdetection reagents include, but are not limited to radiolabelled probes,enzymatic labeled probes (horse radish peroxidase, alkalinephosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin).

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, and the like), and containers which contain thereagents used to detect the hybridized probe, bound antibody, amplifiedproduct, or the like. One skilled in the art will readily recognize thatthe nucleic acid probes described in the present invention can readilybe incorporated into one of the established kit formats which are wellknown in the art.

IV. DNA Constructs Comprising a PYK2 Nucleic Acid Molecule and CellsContaining These Constructs.

The present invention also relates to a recombinant DNA moleculecomprising, 5' to 3', a promoter effective to initiate transcription ina host cell and the above-described nucleic acid molecules. In addition,the present invention relates to a recombinant DNA molecule comprising avector and an above-described nucleic acid molecule. The presentinvention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complimentary toan RNA sequence encoding an amino acid sequence corresponding to theabove-described polypeptide, and a transcriptional termination regionfunctional in said cell. The above-described molecules may be isolatedand/or purified DNA molecules.

The present invention also relates to a cell or organism that containsan above-described nucleic acid molecule. The peptide may be purifiedfrom cells which have been altered to express the peptide. A cell issaid to be "altered to express a desired peptide" when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce or which the cell normally produces at lower levels.One skilled in the art can readily adapt procedures for introducing andexpressing either genomic, cDNA, or synthetic sequences into eithereukaryotic or prokaryotic cells.

A nucleic acid molecule, such as DNA, is said to be "capable ofexpressing" a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are "operably linked" to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5'-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3' to the sequence encoding an PYK2gene may be obtained by the above-described methods. This region may beretained for its transcriptional termination regulatory sequences, suchas termination and polyadenylation. Thus, by retaining the 3'-regionnaturally contiguous to the DNA sequence encoding an PYK2 gene, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3' region functional in the host cellmay be substituted.

Two DNA sequences (such as a promoter region sequence and an PYK2sequence) are said to be operably linked if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of an PYK2 gene sequence, or(3) interfere with the ability of the an PYK2 gene sequence to betranscribed by the promoter region sequence. Thus, a promoter regionwould be operably linked to a DNA sequence if the promoter were capableof effecting transcription of that DNA sequence. Thus, to express anPYK2 gene, transcriptional and translational signals recognized by anappropriate host are necessary.

The present invention encompasses the expression of the PYK2 gene (or afunctional derivative thereof) in either prokaryotic or eukaryoticcells. Prokaryotic hosts are, generally, very efficient and convenientfor the production of recombinant proteins and are, therefore, one typeof preferred expression system for the PYK2 gene. Prokaryotes mostfrequently are represented by various strains of E. coli. However, othermicrobial strains may also be used, including other bacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119 and the like; suitable phage or bacteriophage vectors mayinclude γgt10, γgt11 and the like; and suitable virus vectors mayinclude pMAM-neo, pKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coil, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However,under such conditions, the peptide will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

To express PYK2 (or a functional derivative thereof) in a prokaryoticcell, it is necessary to operably link the PYK2 sequence to a functionalprokaryotic promoter. Such promoters may be either constitutive or, morepreferably, regulatable (i.e., inducible or derepressible). Examples ofconstitutive promoters include the int promoter of bacteriophage λ, thebla promoter of the β-lactamase gene sequence of pBR322, and the CATpromoter of the chloramphenicol acetyl transferase gene sequence ofpPR325, and the like. Examples of inducible prokaryotic promotersinclude the major right and left promoters of bacteriophage λ (P_(L) andP_(R)), the trp, recA, lacZ, lacI, and gal promoters of E. coli, theα-amylase (Ulmanen et at., J. Bacteriol. 162:176-182(1985)) and theζ-28-specific promoters of B. subtilis (Gilman et at., Gene sequence32:11-20(1984)), the promoters of the bacteriophages of Bacillus(Gryczan, In: The Molecular Biology of the Bacilli, Academic Press,Inc., New York (1982)), and Streptomyces promoters (Ward et at., Mol.Gen. Genet. 203:468-478(1986)). Prokaryotic promoters are reviewed byGlick (J. Ind. Microbiot. 1:277-282(1987)); Cenatiempo (Biochimie68:505-516(1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et at.(Ann. Rev. Microbiol. 35:365-404(1981)). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene. As usedherein, "cell", "cell line", and "cell culture" may be usedinterchangeably and all such designations include progeny. Thus, thewords "transformants" or "transformed cells" include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the PYK2 peptide of interest. Suitable hostsmay often include eukaryotic cells. Preferred eukaryotic hosts include,for example, yeast, fungi, insect cells, mammalian cells either in vivo,or in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO or CHO-K1,or cells of lymphoid origin and their derivatives. Preferred mammalianhost cells include SP2/0 and J558L, as well as neuroblastoma cell linessuch as IMR 332 which may provide better capacities for correctpost-translational processing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used. Rubin,Science 240:1453-1459(1988). Alternatively, baculovirus vectors can beengineered to express large amounts of PYK2 in insects cells (Jasny,Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986),Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes areproduced in large quantities when yeast are grown in mediums rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational peptide modifications.A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids which can beutilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Fora mammalian host, several possible vector systems are available for theexpression of PYK2.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of PYK2 in eukaryotic hosts requires the use of eukaryoticregulatory regions. Such regions will, in general, include a promoterregion sufficient to direct the initiation of RNA synthesis. Preferredeukaryotic promoters include, for example, the promoter of the mousemetallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288(1982)); the TK promoter of Herpes virus (McKnight, Cell31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310(1981)); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975(1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes PYK2 (or a functional derivative thereof) does not contain anyintervening codons which are capable of encoding a methionine (i.e.,AUG). The presence of such codons results either in a formation of afusion protein (if the AUG codon is in the same reading frame as thePYK2 coding sequence) or a frame-shift mutation (if the AUG codon is notin the same reading frame as the PYK2 coding sequence).

A PYK2 nucleic acid molecule and an operably linked promoter may beintroduced into a recipient prokaryotic or eukaryotic cell either as anonreplicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the gene may occur through the transient expression of the introducedsequence. Alternatively, permanent expression may occur through theintegration of the introduced DNA sequence into the host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, Molec. Cell. Biol.3:280(1983).

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to"shuttle" the vector between host cells of different species. Preferredprokaryotic vectors include plasmids such as those capable ofreplication in E. Coli (such as, for example, pBR322, ColEl, pSC101,pACYC 184, πVX. Such plasmids are, for example, disclosed by Sambrook(cf. Molecular Cloning: A Laboratory Manual, second edition, edited bySambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)).Bacillus plasmids include pC194, pC221, pT127, and the like. Suchplasmids are disclosed by Gryczan (In: The Molecular Biology of theBacitli, Academic Press, New York (1982), pp. 307-329). SuitableStreptomyces plasmids include p1J101 (Kendall et al., J. Bacteriol.169:4177-4183 (1987)), and streptomyces bacteriophages such as φC31(Chater et al., In: Sixth International Symposium on ActinomycetalesBiology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54).Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis.8:693-704(1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp.19:265-274(1982); Broach, In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell28:203-204 (1982); Bollon et at., J. Ctin. Hematol. Oncol. 10:39-48(1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,Gene Sequence Expression, Academic Press, New York, pp. 563-608(1980).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofPYK2 or fragments thereof. This can take place in the transformed cellsas such, or following the induction of these cells to differentiate (forexample, by administration of bromodeoxyuracil to neuroblastoma cells orthe like). A variety of incubation conditions can be used to form thepeptide of the present invention. The most preferred conditions arethose which mimic physiological conditions.

V. Purified PYK2 Polypeptides.

A variety of methodologies known in the art can be utilized to obtainthe peptide of the present invention. The peptide may be purified fromtissues or cells which naturally produce the peptide. Alternatively, theabove-described isolated nucleic acid fragments could be used to expressthe PYK2 protein in any organism. The samples of the present inventioninclude cells, protein extracts or membrane extracts of cells, orbiological fluids. The sample will vary based on the assay format, thedetection method and the nature of the tissues, cells or extracts usedas the sample.

Any eukaryotic organism can be used as a source for the peptide of theinvention, as long as the source organism naturally contains such apeptide. As used herein, "source organism" refers to the originalorganism from which the amino acid sequence of the subunit is derived,regardless of the organism the subunit is expressed in and ultimatelyisolated from.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain the peptide free of natural contaminants.These include, but are not limited to: size-exclusion chromatography,HPLC, ion-exchange chromatography, and immuno-affinity chromatography.

VI. PYK2 Antibody And Hybridoma.

The present invention relates to an antibody having binding affinity toa PYK2 polypeptide. The polypeptide may have the amino acid sequence setforth in SEQ ID NO:1, or mutant or species variation thereof, or atleast 9 contiguous amino acids thereof (preferably, at least 10, 15, 20,or 30 contiguous amino acids thereof).

The present invention also relates to an antibody having specificbinding affinity to an PYK2 polypeptide. Such an antibody may beisolated by comparing its binding affinity to a PYK2 polypeptide withits binding affinity to another polypeptide. Those which bindselectively to PYK2 would be chosen for use in methods requiring adistinction between PYK2 and other polypeptides. Such methods couldinclude, but should not be limited to, the analysis of altered PYK2expression in tissue containing other polypeptides such as FAK.

The PYK2 proteins of the present invention can be used in a variety ofprocedures and methods, such as for the generation of antibodies, foruse in identifying pharmaceutical compositions, and for studyingDNA/protein interaction.

The PYK2 peptide of the present invention can be used to produceantibodies or hybridomas. One skilled in the art will recognize that ifan antibody is desired, such a peptide would be generated as describedherein and used as an immunogen. The antibodies of the present inventioninclude monoclonal and polyclonal antibodies, as well fragments of theseantibodies, and humanized forms. Humanized forms of the antibodies ofthe present invention may be generated using one of the procedures knownin the art such as chimerization or CDR grafting. The present inventionalso relates to a hybridoma which produces the above-describedmonoclonal antibody, or binding fragment thereof. A hybridoma is animmortalized cell line which is capable of secreting a specificmonoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, "Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology," Elsevier Science Publishers, Amsterdam, The Netherlands(1984); St. Groth et al., J. Immunol. Methods 35:1-21(1980)). Any animal(mouse, rabbit, and the like) which is known to produce antibodies canbe immunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Res. 175:109-124(1988)). Hybridomas secreting the desiredantibodies are cloned and the class and subclass is determined usingprocedures known in the art (Campbell, Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology, supra(1984)).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures. Theabove-described antibodies may be detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art, for example, see (Stemberger et al., J. Histochem. Cytochem.18:315(1970); Bayer et al., Meth. Enzym. 62:308(1979); Engval et al.,Immunot. 109:129(1972); Goding, J. Immunol. Meth. 13:215(1976)). Thelabeled antibodies of the present invention can be used for in vitro, invivo, and in situ assays to identify cells or tissues which express aspecific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., "Handbook of Experimental Immunology" 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10(1986); Jacoby etal., Meth. Enzym. 34 Academic Press, New York (1974)). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,"Application of Synthetic Peptides: Antisense Peptides", In SyntheticPeptides, A User's Guide, W. H. Freeman, N.Y., pp. 289-307(1992), andKaspczak et al., Biochemistry 28:9230-8(1989).

Anti-peptide peptides can be generated by replacing the basic amino acidresidues found in the PYK2 peptide sequence with acidic residues, whilemaintaining hydrophobic and uncharged polar groups. For example, lysine,arginine, and/or histidine residues are replaced with aspartic acid orglutamic acid and glutamic acid residues are replaced by lysine,arginine or histidine.

VII. An Antibody Based Method And Kit For Detecting PYK2.

The present invention encompasses a method of detecting an PYK2polypeptide in a sample, comprising: a) contacting the sample with anabove-described antibody, under conditions such that immunocomplexesform, and b) detecting the presence of said antibody bound to thepolypeptide. In detail, the methods comprise incubating a test samplewith one or more of the antibodies of the present invention and assayingwhether the antibody binds to the test sample. Altered levels of PYK2 ina sample as compared to normal levels may indicate muscular disease.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, "An Introduction to Radioimmunoassay andRelated Techniques" Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., "Techniques in Immunocytochemistry,"Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, "Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology," Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily adaptedin order to obtain a sample which is capable with the system utilized.

A kit contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: i) a firstcontainer means containing an above-described antibody, and ii) secondcontainer means containing a conjugate comprising a binding partner ofthe antibody and a label. In another preferred embodiment, the kitfurther comprises one or more other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting thepresence of bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody.

The compartmentalized kit may be as described above for nucleic acidprobe kits. One skilled in the art will readily recognize that theantibodies described in the present invention can readily beincorporated into one of the established kit formats which are wellknown in the art.

VIII. Isolation of Compounds Which Interact With PYK2.

The present invention also relates to a method of detecting a compoundcapable of binding to a PYK2 polypeptide comprising incubating thecompound with PYK2 and detecting the presence of the compound bound toPYK2. The compound may be present within a complex mixture, for example,serum, body fluid, or cell extracts.

The present invention also relates to a method of detecting an agonistor antagonist of PYK2 activity comprising incubating cells that producePYK2 in the presence of a compound and detecting changes in the level ofPYK2 activity. The compounds thus identified would produce a change inactivity indicative of the presence of the compound. The compound may bepresent within a complex mixture, for example, serum, body fluid, orcell extracts. Once the compound is identified it can be isolated usingtechniques well known in the art.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing PYK2 associated activity in a mammalcomprising administering to said mammal an agonist or antagonist to PYK2in an amount sufficient to effect said agonism or antagonism. A methodof treating diabetes mellitus, skeletal muscle diseases, Alzheimer'sdisease, or peripheral neuropathies in a mammal with an agonist orantagonist of PYK2 activity comprising administering the agonist orantagonist to a mammal in an amount sufficient to agonize or antagonizePYK2 associated functions is also encompassed in the presentapplication.

IX. Transgenic Animals.

A variety of methods are available for the production of transgenicanimals associated with this invention. DNA can be injected into thepronucleus of a fertilized egg before fusion of the male and femalepronuclei, or injected into the nucleus of an embryonic cell (e.g., thenucleus of a two-cell embryo) following the initiation of cell division(Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985) ) .Embryos can be infected with viruses, especially retroviruses, modifiedto carry inorganic-ion receptor nucleotide sequences of the invention.

Pluripotent stem cells derived from the inner cell mass of the embryoand stabilized in culture can be manipulated in culture to incorporatenucleotide sequences of the invention. A transgenic animal can beproduced from such cells through implantation into a blastocyst that isimplanted into a foster mother and allowed to come to term. Animalssuitable for transgenic experiments can be obtained from standardcommercial sources such as Charles River (Wilmington, Mass.), Taconic(Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), etc.

The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia 47: 897-905 (1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

By way of example only, to prepare a transgenic mouse, female mice areinduced to superovulate. Females are placed with males, and the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer etal., Cell 63:1099-1112 (1990).

Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press (1987).

In cases involving random gene integration, a clone containing thesequence(s) of the invention is cotransfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination. Capecchi,Science 244: 1288-1292 (1989). Methods for positive selection of therecombination event (i.e., neo resistance) and dual positive-negativeselection (i.e., neo resistance and gancyclovir resistance) and thesubsequent identification of the desired clones by PCR have beendescribed by Capecchi, supra and Joyner et al., Nature 338: 153-156(1989), the teachings of which are incorporated herein. The final phaseof the procedure is to inject targeted ES cells into blastocysts and totransfer the blastocysts into pseudopregnant females. The resultingchimeric animals are bred and the offspring are analyzed by Southernblotting to identify individuals that carry the transgene. Proceduresfor the production of non-rodent mammals and other animals have beendiscussed by others. See Houdebine and Chourrout, supra; Pursel et al.,Science 244:1281-1288 (1989); and Simms et al., Bio/Technology 6:179-183(1988).

X. Compositions

The present invention relates to removing or reducing an abnormality ina signal transduction pathway, wherein the signal transduction pathwaycontains a PYK2 polypeptide. The present invention also relates tocompositions and methods for the treatment of disorders which involvemodulating the activity and/or level of individual components, andrelates to methods for the identification of agents for such treatments.Additionally, the present invention relates to methods and compositionsfor prognostic evaluation of such disorders.

Described herein are compositions and methods for the prevention,prognostic evaluation, and treatment of disorders described herein,preferably cell proliferative disorders and hematopoietic celldisorders, in which a PYK2 polypeptide may be involved.

First, methods and compositions for the treatment of such disorders aredescribed. Such methods and compositions may include, but are notlimited to the agents capable of decreasing or inhibiting theinteraction between a PYK2 polypeptide and a PYK2 polypeptide bindingpartner and agents capable of inhibiting or decreasing the activity ofsuch complexes, agents capable of modulating the activity and/or levelof individual components of the proteins, and the use and administrationof such agents. Agents capable of modulating the activity and/or levelof interaction between a PYK2 polypeptide and a PYK2 polypeptide bindingpartner include those agents that inhibit or decrease thedephosphorylating activity of tyrosine phosphatases.

Second, methods are described for the identification of such agents.These methods may include, for example, assays to identify agentscapable of disrupting or inhibiting or promoting the interaction betweencomponents of the complexes (e.g., PYK2:NBP complexes), and may alsoinclude paradigms and strategies for the rational design of drugscapable of disruption and/or inhibition and/or promotion of suchcomplexes.

The complexes involved in the invention include a PYK2 polypeptide and aNBP or derivatives thereof, as described below. Under standardphysiological conditions, the components of such complexes are capableof forming stable, non-covalent attachments with one or more of theother complex components. Methods for the purification and production ofsuch protein complexes, and of cells that exhibit such complexes aredescribed below.

XI. Disruption of Protein Complexes

Disruption of complexes (e.g., PYK2:NBP complexes), for example bydecreasing or inhibiting the interactions between component members ofsuch a complex may have differing modulatory effects on the eventinvolved, depending on the individual protein complex. "Disruption", asused here, is meant to refer not only to a physical separation ofprotein complex components, but also refers to a perturbation of theactivity of the complexes, regardless of whether or not such complexesremain able, physically, to form. "Activity", as used here, refers tothe function of the protein complex in the signal transduction cascadeof the cell in which such a complex is formed, i.e., refers to thefunction of the complex in effecting or inhibiting a transduction of anextracellular signal into a cell. For example, the effect of complexdisruption may augment, reduce, or block a signal normally transducedinto the cell. Likewise, depending on the disorder involved, eitheraugmentation, reduction, or blockage of a signal normally transducedinto the cell will be desirable for the treatment of the disorder.

A disorder involving a complex may, for example, develop because thepresence of such a complex brings about the aberrant inhibition of anormal signal transduction event. In such a case, the disruption of thecomplex would allow the restoration of the usual signal transductionevent. Further, an aberrant complex may bring about an alteredsubcellular adapter protein localization, which may result in, forexample, dysfunctional cellular events. An inhibition of the complex inthis case would allow for restoration or maintenance of a normalcellular architecture. Still further, an agent or agents that cause(s)disruption of the complex may bring about the disruption of theinteractions among other potential components of a complex.

Nucleotide sequences encoding peptide agents which are to be utilizedintracellularly may be expressed in the cells of interest, usingtechniques which are well known to those of ordinary skill in the art.For example, expression vectors derived from viruses such asretroviruses, vaccinia virus, adenoviruses, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery andexpression of such nucleotide sequences into the targeted cellpopulation. Methods for the construction of such vectors are well known.See, for example, the techniques described in Maniatis et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y. and in Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y. 1989.Complex-binding domains can be identified using, for example, techniquessuch as those described in Rotin et al. (Rotin et al., EMBO J.11:559-567, 1992), Songyang et al. (Songyang et al., Cell 72:767-778,1993), Felder et al., Mol. Cell. Biol. 13:1449-1455, 1993), Fantl et al.(Cell 69:413-422, 1992), and Domchek et al. (Biochemistry 31:9865-9870,1992).

Alternatively, antibodies capable of interfering with complex formationmay be produced as described below and administered for the treatment ofdisorders involving a component capable of forming a complex withanother protein. Alternatively, nucleotide sequences encodingsingle-chain antibodies may be expressed within the target cellpopulation by utilizing, for example, techniques such as those describedin Marasco et al. (Marasco et al., Proc. Natl. Acad. Sci. USA90:7889-7893, 1993). Agents which act intracellularly to interfere withthe formation and/or activity of the protein complexes of the inventionmay also be small organic or inorganic compounds. A method foridentifying these and other intracellular agents is described below.

XII. Antibodies to Complexes

Described herein are methods for the production of antibodies which arecapable of specifically recognizing a complex or an epitope thereof, orof specifically recognizing an epitope on either of the components ofthe complex, especially those epitopes which would not be recognized bythe antibody when the component is present separate and apart from thecomplex. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments,fragments produced by a FAb expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a complex in abiological sample, or, alternatively, as a method for the inhibition ofa complex formation, thus inhibiting the development of a disorder.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a complex, or an antigenic functional derivative thereof. Forthe production of polyclonal antibodies, various host animals may beimmunized by injection with the complex including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

A monoclonal antibody, which is a substantially homogeneous populationof antibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to thehybridoma technique of Kohler and Milstein (Nature 256:495-497, 1975)and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl.Acad. Sci. USA 80:2026-2030, 1983), and the EBV-hybridoma technique(Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss,Inc,.1985, pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

In addition, techniques developed for the production of "chimericantibodies" (Morrison et al., Proc. Natl. Acad. Sci., 81:6851-6855,1984; Neuberger et al., Nature, 312:604-608, 1984; Takeda et al.,Nature, 314:452-454, 1985) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. 4,946,778; Bird, Science 242:423-426, 1988; Hustonet al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Ward et al.,Nature 334:544-546, 1989) can be adapted to produce complex-specificsingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragment of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

Antibody fragments which contain specific binding sites of a complex maybe generated by known techniques. For example, such fragments includebut are not limited to: the F(ab')₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab')₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityto the PTK/adapter complex.

One or more components of a protein complex may be present at a higherthan normal cellular level (i.e., higher than the concentration known tousually be present in the cell type exhibiting the protein complex ofinterest and/or may exhibit an abnormally increased level of cellularactivity (i.e., greater than the activity known to usually be present inthe cell type exhibiting the protein complex of interest).

For example, the gene encoding a protein complex component may begin tobe overexpressed, or may be amplified (i.e., its gene copy number may beincreased) in certain cells, leading to an increased number of componentmolecules within these cells. Additionally, a gene encoding a proteincomplex component may begin to express a modified protein product thatexhibits a greater than normal level of activity. "Activity", here,refers to the normal cellular function of the component, eitherenzymatic or structural whose function may include, for example,bringing two or more cellular molecules into the appropriate proximity.

Such an increase in the cellular level and/or activity of a proteincomplex may lead to the development of a disorder. Treatment of suchdisorders may, therefore, be effectuated by the administration of agentswhich decrease the cellular level and/or the activity of theoverexpressed and/or overactive protein complex component. Techniquesfor decreasing the cellular level and/or the activity of one or more ofthe protein complex components of interest may include, but are notlimited to antisense or ribozyme approaches, and/or gene therapyapproaches, each of which is well known to those of skill in the art.

XIII. Antisense and Ribozyme Approaches

Included in the scope of the invention are oligoribonucleotides,including antisense RNA and DNA molecules and ribozymes that function toinhibit translation of one or more components of a protein complex.Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by binding to targeted mRNA and preventing protein translation.With respect to antisense DNA, oligodeoxyribonucleotides derived fromthe translation initiation site, e.g., between -10 and +10 regions ofthe relevant nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific interaction of the ribozyme molecule to complementary targetRNA, followed by a endonucleolytic cleavage. Within the scope of theinvention are engineered hammerhead or other motif ribozyme moleculesthat specifically and efficiently catalyze endonucleolytic cleavage ofRNA sequences encoding protein complex components.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatetargets may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays. See, Draper PCT WO 93/23569.

Both anti-sense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of RNAmolecules. See, Draper, id. hereby incorporated by reference herein.These include techniques for chemically synthesizingoligodeoxyribonucleotides well known in the art such as for examplesolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a meansof increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of themolecule or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

XIV. Gene Therapy

PYK2 or its genetic sequences will also be useful in gene therapy(reviewed in Miller, Nature 357:455-460, (1992). Miller states thatadvances have resulted in practical approaches to human gene therapythat have demonstrated positive initial results. An in vivo model ofgene therapy for human severe combined immunodeficiency is described inFerrari, et al., Science 251:1363-1366, (1991). The basic science ofgene therapy is described in Mulligan, Science 260:926-931, (1993).

In one preferred embodiment, an expression vector containing the PYK2coding sequence is inserted into cells, the cells are grown in vitro andthen infused in large numbers into patients. In another preferredembodiment, a DNA segment containing a promoter of choice (for example astrong promoter) is transferred into cells containing an endogenous PYK2in such a manner that the promoter segment enhances expression of theendogenous PYK2 gene (for example, the promoter segment is transferredto the cell such that it becomes directly linked to the endogenous PYK2gene).

The gene therapy may involve the use of an adenovirus containing PYK2cDNA targeted to a tumor, systemic PYK2 increase by implantation ofengineered cells, injection with PYK2 virus, or injection of naked PYK2DNA into appropriate tissues.

Target cell populations (e.g., hematopoietic or nerve cells) may bemodified by introducing altered forms of PYK2 in order to modulate theactivity of such cells. For example, by reducing or inhibiting an anerve cell within target cells, an abnormal response leading to acondition may be decreased, inhibited, or reversed. Deletion or missensemutants of PYK2, that retain the ability to interact with othercomponents of the nervous system but cannot participate in normalfunction may be used to inhibit an abnormal, deleterious response.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, several RNAviruses, or bovine papilloma virus, may be used for delivery ofnucleotide sequences (e.g., cDNA) encoding recombinant PYK2 protein intothe targeted cell population (e.g., tumor cells). Methods which are wellknown to those skilled in the art can be used to construct recombinantviral vectors containing coding sequences. See, for example, thetechniques described in Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. (1989), and in Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. (1989). Alternatively,recombinant nucleic acid molecules encoding protein sequences can beused as naked DNA or in reconstituted system e.g., liposomes or otherlipid systems for delivery to target cells (See e.g., Felgner et al.,Nature 337:387-8, 1989). Several other methods for the direct transferof plasmid DNA into cells exist for use in human gene therapy andinvolve targeting the DNA to receptors on cells by complexing theplasmid DNA to proteins. See, Miller, supra.

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell, through a process ofmicroinjection. Capecchi MR, Cell 22:479-88 (1980). Once recombinantgenes are introduced into a cell, they can be recognized by the cellsnormal mechanisms for transcription and translation, and a gene productwill be expressed. Other methods have also been attempted forintroducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO₄ and taken intocells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52(1987)) ; electroporation, wherein cells are exposed to large voltagepulses to introduce holes into the membrane (Chu G. et al., NucleicAcids Res., 15:1311-26 (1987)); lipofection/liposome fusion, wherein DNAis packaged into lipophilic vesicles which fuse with a target cell(Felgner PL., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7 (1987)); andparticle bombardment using DNA bound to small projectiles (Yang NS. etal., Proc. Natl. Acad. Sci. 87:9568-72 (1990)). Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins.

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene. Curiel DT et al., Am. J. Respir.Cell. Mol. Biol., 6:247-52 (1992).

As used herein "gene transfer" means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

As used herein "gene therapy" is a form of gene transfer and is includedwithin the definition of gene transfer as used herein and specificallyrefers to gene transfer to express a therapeutic product from a cell invivo or in vitro. Gene transfer can be performed ex vivo on cells whichare then transplanted into a patient, or can be performed by directadministration of the nucleic acid or nucleic acid-protein complex intothe patient.

In another preferred embodiment, a vector having nucleic acid sequencesencoding PYK2 is provided in which the nucleic acid sequence isexpressed only in specific tissue. Methods of achieving tissue-specificgene expression as set forth in International Publication No. WO93/09236, filed Nov. 3, 1992 and published May 13, 1993.

In all of the preceding vectors set forth above, a further aspect of theinvention is that the nucleic acid sequence contained in the vector mayinclude additions, deletions or modifications to some or all of thesequence of the nucleic acid, as defined above.

In another preferred embodiment, a method of gene replacement is setforth. "Gene replacement" as used herein means supplying a nucleic acidsequence which is capable of being expressed in vivo in an animal andthereby providing or augmenting the function of an endogenous gene whichis missing or defective in the animal.

XV. Pharmaceutical Formulations and Modes of Administration

The particular compound, antibody, antisense or ribozyme molecule thataffects the protein complexes and the disorder of interest can beadministered to a patient either by themselves, or in pharmaceuticalcompositions where it is mixed with suitable carriers or excipient(s).In treating a patient exhibiting a disorder of interest, atherapeutically effective amount of a agent or agents such as these isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal inhibition of the cellular level and/oractivity of a complex component). Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity) The magnitude of anadministrated dose in the management of the oncogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/orpolyvinylpyrrolidone (PVP) . If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coating. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Some methods of delivery that may be used include:

a. encapsulation in liposomes,

b. transduction by retroviral vectors,

c. localization to nuclear compartment utilizing nuclear targeting sitefound on most nuclear proteins,

d. transfection of cells ex vivo with subsequent reimplantation oradministration of the transfected cells,

e. a DNA transporter system.

A PYK2 nucleic acid sequence may be administered utilizing an ex vivoapproach whereby cells are removed from an animal, transduced with thePYK2 nucleic acid sequence and reimplanted into the animal. The livercan be accessed by an ex vivo approach by removing hepatocytes from ananimal, transducing the hepatocytes in vitro with the PYK2 nucleic acidsequence and reimplanting them into the animal (e.g., as described forrabbits by Chowdhury et al, Science 254: 1802-1805, 1991, or in humansby Wilson, Hum. Gene Ther. 3: 179-222, 1992) incorporated herein byreference.

Many nonviral techniques for the delivery of a PYK2 nucleic acidsequence into a cell can be used, including direct naked DNA uptake(e.g., Wolff et al., Science 247: 1465-1468, 1990), receptor-mediatedDNA uptake, e.g., using DNA coupled to asialoorosomucoid which is takenup by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol.Chem. 262: 4429-4432, 1987; Wu et al., J. Biol. Chem. 266: 14338-14342,1991), and liposome-mediated delivery (e.g., Kaneda et al., Expt. CellRes. 173: 56-69, 1987; Kaneda et al., Science 243: 375-378, 1989; Zhu etal., Science 261: 209-211, 1993). Many of these physical methods can becombined with one another and with viral techniques; enhancement ofreceptor-mediated DNA uptake can be effected, for example, by combiningits use with adenovirus (Curiel et al., Proc. Natl. Acad. Sci. USA 88:8850-8854, 1991; Cristiano et al., Proc. Natl. Acad. Sci. USA 90:2122-2126, 1993).

The PYK2 or nucleic acid encoding PYK2 may also be administered via animplanted device that provides a support for growing cells. Thus, thecells may remain in the implanted device and still provide the usefuland therapeutic agents of the present invention.

XVI. Identification of Agents

The complexes, components of such complexes, functional equivalentsthereof, and/or cell lines that express such components and exhibit suchprotein complexes may be used to screen for additional compounds,antibodies, or other molecules capable of modulating the signaltransduction event such complexes are involved in. Methods for purifyingand/or producing such complexes, components of the complexes, functionalequivalents thereof, and/or cell lines are described herein. Thecompounds, antibodies, or other molecules identified may, for example,act to disrupt the protein complexes of the invention (i.e., decrease orinhibit interactions between component members of the complexes, therebycausing physical separation of the components, and/or perturbing theactivity of the complexes) or may lower the cellular level and/ordecrease the activity of one or more of the components of suchcomplexes.

Such compounds may include, but are not limited to, peptides made of D-and/or L-configuration amino acids (in, for example, the form of randompeptide libraries; see Lam et al., Nature 354:82-84, 1991),phosphopeptides (in, for example, the form of random or partiallydegenerate, directed phosphopeptide libraries, see Songyang et al., Cell767-778, 1993), antibodies, and small organic or inorganic molecules.Synthetic compounds, natural products, and other sources of potentiallybiologically active materials may be screened in a variety of ways, asdescribed herein. The compounds, antibodies, or other moleculesidentified may be used as oncogenic disorder treatments, as describedherein.

Compounds that bind to individual components, or functional portions ofthe individual components of the complexes (and may additionally becapable of disrupting complex formation) may be identified.

One such method included within the scope of the invention is a methodfor identifying an agent to be tested for an ability to modulate asignal transduction pathway disorder. The method involves exposing atleast one agent to a protein comprising a functional portion of a memberof the protein complex for a time sufficient to allow binding of theagent to the functional portion of the member; removing non-boundagents; and determining the presence of the compound bound to thefunctional portion of the member of the protein complex, therebyidentifying an agent to be tested for an ability to modulate a disorderinvolving a polypeptide complex.

By "signal transduction disorder" is meant any disease or conditionassociated with an abnormality in a signal transduction pathway. Theprotein complex referred to below is a physical association of dynaminand a PYK2 polypeptide. The level of interaction between the twocomponents of the complex may be abnormal and thus cause the abnormalityin the signal transduction pathway. Alternatively, the level ofinteraction between the complex components may be normal, but affectingthat interaction may effectively treat a signal transduction pathwaydisorder.

The term "protein" refers to a compound formed of 5-50 or more aminoacids joined together by peptide bonds. An "amino acid" is a subunitthat is polymerized to form proteins and there are twenty amino acidsthat are universally found in proteins. The general formula for an aminoacid is H₂ N--CHR--COOH, in which the R group can be anything from ahydrogen atom (as in the amino acid glycine) to a complex ring (as inthe amino acid tryptophan).

A functional portion of an individual component of the complexes may bedefined here as a protein portion of an individual component of acomplex still capable of forming a stable complex with another member ofthe complex under standard cellular and physiological conditions. Forexample, a functional portion of a component may include, but is notlimited to, a protein portion of dynamin which is still capable ofstably binding a corresponding PYK2 polypeptide of an associatedprotein, and thus is still capable of forming a complex with thatprotein. Further, in the case of the catalytic domains of the individualcomponents of the invention, a functional portion of a catalytic domainmay refer to a protein still capable of stably binding a substratemolecule under standard physiological conditions.

One method utilizing this approach that may be pursued in the isolationof such complex component-binding molecules would include the attachmentof a component molecule, or a functional portion thereof, to a solidmatrix, such as agarose or plastic beads, microtiter wells, petridishes, or membranes composed of, for example, nylon or nitrocellulose,and the subsequent incubation of the attached component molecule in thepresence of a potential component-binding compound or compounds.Attachment to said solid support may be direct or by means of acomponent specific antibody bound directly to the solid support. Afterincubation, unbound compounds are washed away, component-bound compoundsare recovered. By utilizing this procedure, large numbers of types ofmolecules may be simultaneously screened for complex component-bindingactivity.

The complex components which may be utilized in the above screeningmethod may include, but are not limited to, molecules or functionalportions thereof, such as catalytic domains, phosphorylation domains,extracellular domains, or portions of extracellular domains, such asligand-binding domains, and adaptor proteins, or functional portionsthereof. The peptides used may be phosphorylated, e.g., may contain atleast one phosphorylated amino acid residue, preferably a phosphorylatedTyr amino acid residue, or may be unphosphorylated. A phosphorylationdomain may be defined as a peptide region that is specificallyphosphorylated at certain amino acid residues. A functional portion ofsuch a phosphorylation domain may be defined as a peptide capable ofbeing specifically phosphorylated at certain amino acids by a specificprotein.

Molecules exhibiting binding activity may be further screened for anability to disrupt protein complexes. Alternatively, molecules may bedirectly screened for an ability to promote the complexes. For example,in vitro complex formation may be assayed by, first, immobilizing onecomponent, or a functional portion thereof, of the complex of interestto a solid support. Second, the immobilized complex component may beexposed to a compound such as one identified as above, and to the secondcomponent, or a functional portion thereof, of the complex of interest.Third, it may be determined whether or not the second component is stillcapable of forming a complex with the immobilized component in thepresence of the compound. In addition, one could look for an increase inbinding.

Additionally, complex formation in a whole cell may be assayed byutilizing co-immunoprecipitation techniques well known to those of skillin the art. Briefly, a cell line capable of forming a complex ofinterest may be exposed to a compound such as one identified as above,and a cell lysate may be prepared from this exposed cell line. Anantibody raised against one of the components of the complex of interestmay be added to the cell lysate, and subjected to standardimmunoprecipitation techniques. In cases where a complex is stillformed, the immunoprecipitation will precipitate the complex, whereas incases where the complex has been disrupted, only the complex componentto which the antibody is raised will be precipitated.

A preferred method for assessing modulation of complex formation withina cell utilizes a method similar to that described above. Briefly, acell line capable of forming a complex of interest is exposed to a testcompound. The cells are lysed and the lysate contacted with an antibodyspecific to one component of the complex, said antibody having beenpreviously bound to a solid support. Unbound material is washed away,and the bound material is exposed to a second antibody, said secondantibody binding specifically to a second component of the complex. Theamount of second antibody bound is easily detected by techniques wellknown in the art. Cells exposed to an inhibitory test compound will haveformed a lesser amount of complex compared to cells not exposed to thetest compound, as measured by the amount of second antibody bound. Cellsexposed to a test compound that promotes complex formation will have anincreased amount of second antibody bound.

The effect of an agent on the differentiation capability of the complexof interest may be directly assayed. Such agents may, but are notrequired to, include those agents identified by utilizing the abovescreening technique. For example, an agent or agents may be administeredto a cell such as a neuronal cell, capable of forming a complex, forexample, which, in the absence of any agent, would not lead to thecell's differentiation. The differentiation state of the cell may thenbe measured either in vitro or in vivo. One method of measurement mayinvolve observing the amount of neurile growth present.

Agents capable of disrupting complex formation and capable of reducingor inhibiting disorders, which involve the formation of such complexes,or which involve the lack of formation of such complexes, may be used inthe treatment of patients exhibiting or at risk for such disorders. Asufficient amount of agent or agents such as those described above maybe administered to a patient so that the symptoms of the disease orcondition are reduced or eliminated.

XVII. Purification and Production of Complexes

Described in this Section are methods for the synthesis or recombinantexpression of components, or fragments thereof, of the protein complexesof the invention. Also described herein are methods by which cellsexhibiting the protein complexes of the invention may be engineered.

The complexes of the invention may be substantially purified, i.e., maybe purified away from at least 90% (on a weight basis), and from atleast 99%, if desired, of other proteins, glycoproteins, and othermacromolecules with which it is associated. Such purification can beachieved by utilizing a variety of procedures well known to those ofskill in the art, such as subjecting cells, tissue or fluid containingthe complex to a combination of standard methods, for example, ammoniumsulfate precipitation, molecular sieve chromatography, and/or ionexchange chromatography.

Alternatively, or additionally, a complex may be purified byimmunoaffinity chromatography using an immunoabsorbent column to whichan antibody is immobilized which is capable of binding to one or morecomponents of the complex. Such an antibody may be monoclonal orpolyclonal in origin. Other useful types of affinity purification forthe protein complex may utilize, for example, a solid-phase substratewhich binds the catalytic kinase domain of a protein, or an immobilizedbinding site for noncatalytic domains of the components of the complex,which bind in such a manner as to not disrupt the complex. The complexof the present invention may be biochemically purified from a variety ofcell or tissue sources.

Methods for the synthesis of polypeptides or fragments thereof, whichare capable of acting as components of the complexes of the presentinvention, are well-known to those of ordinary skill in the art. See,for example, Creighton, Proteins: Structures and Molecular Principles,W. H. Freeman and Co., N.Y. (1983), which is incorporated herein, byreference, in its entirety.

Components of a complex which have been separately synthesized orrecombinantly produced, may be reconstituted to form a complex bystandard biochemical techniques well known to those skilled in the art.For example, samples containing the components of the complex may becombined in a solution buffered with greater than about 150 mM NaCl, ata physiological pH in the range of 7, at room temperature. For example,a buffer comprising 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% glycerol,1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and 2 mM EDTA could beused.

Methods for preparing the components of complexes of the invention byexpressing nucleic acid encoding proteins are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing protein coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.DNA and RNA synthesis may, additionally, be performed using an automatedsynthesizers. See, for example, the techniques described in Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1989), and in Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y. (1989).

A variety of host-expression vector systems may be utilized to expressthe coding sequences of the components of the complexes of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced, but also represent cellswhich may, when transformed or transfected with the appropriatenucleotide coding sequences, exhibit the protein complexes of theinvention. These include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining protein coding sequences; yeast (e.g., Saccharomyces andPichia) transformed with recombinant yeast expression vectors containingthe protein coding sequencers; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing theprotein coding sequences; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing the protein coding sequencescoding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293,3T3) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter)

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the complexbeing expressed. For example, when large quantities of complex proteinsare to be produced for the generation of antibodies or to screen peptidelibraries, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther et al., EMBO J. 2:1791, 1983), in which the proteincoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye and Inouye, Nucleic acids Res. 13:3101-3109, 1985; VanHeeke & Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned proteincan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The complex coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of thePTK/adaptor complex coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (e.g., see Smith et al.,J. Biol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the complex coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing proteins in infected hosts. (E.g., See Logan & Shenk, Proc.Natl. Acad. Sci. USA 81:3655-3659, 1984) Specific initiation signals mayalso be required for efficient translation of inserted coding sequences.These signals include the ATG initiation codon and adjacent sequences.

In cases where an entire protein gene, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of the coding sequence isinserted, exogenous translational control signals, including the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:516-544, 1987)

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably coexpressboth the proteins may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with the protein encoding DNA independently or coordinatelycontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker.

Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which coexpress both thePTK and adaptor protein. Such engineered cell lines are particularlyuseful in screening and evaluation of compounds that effect signalsmediated by the complexes.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., Natl.Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci. USA78:1527, 1981); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,J. Mol. Biol. 150:1, 1981); and hygro, which confers resistance tohygromycin (Santerre et al. Gene 30:147, 1984) genes.

New members of the protein families capable of forming the complexes ofthe invention may be identified and isolated by molecular biologicaltechniques well known in the art. For example, a previously unknownprotein encoding gene may be isolated by performing a polymerase chainreaction (PCR) using two degenerate oligonucleotide primer poolsdesigned on the basis of highly conserved sequences within domainscommon to members of the protein family.

The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from cell lines or tissue known toexpress complexes. The PCR product may be subcloned and sequenced toinsure that the amplified sequences represent the sequences of a memberof the PTK or adaptor subfamily. The PCR fragment may then be used toisolate a full length protein cDNA clone by radioactively labeling theamplified fragment and screening a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary. For a review of cloning strategies which may be used. See e.g.,Maniatis, Molecular Cloning: A Laboratory Manual, Cold Springs HarborPress, N.Y. (1989); and Ausubel et al., Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y.(1989). A general method for cloning previously unknown proteins hasbeen described by Skolnik (Skolnik, E. Y., Cell 65:75, 1991) and Skolniket al., (U.S. patent application Ser. No. 07/643,237) which areincorporated herein, by reference, in their entirety, includingdrawings.

XVIII. Derivatives of Complexes

Also provided herein are functional derivatives of a complex. By"functional derivative" is meant a "chemical derivative," "fragment,""variant," "chimera," or "hybrid" of the complex, which terms aredefined below. A functional derivative retains at least a portion of thefunction of the protein, for example reactivity with an antibodyspecific for the complex, enzymatic activity or binding activitymediated through noncatalytic domains, which permits its utility inaccordance with the present invention.

A "chemical derivative" of the complex contains additional chemicalmoieties not normally a part of the protein. Such moieties may improvethe molecule's solubility, absorption, biological half life, and thelike. The moieties may alternatively decrease the toxicity of themolecule, eliminate or attenuate any undesirable side effect of themolecule, and the like. Moieties capable of mediating such effects aredisclosed in Remington's Pharmaceutical Sciences (1980). Procedures forcoupling such moieties to a molecule are well known in the art. Covalentmodifications of the protein complex or peptides are included within thescope of this invention. Such modifications may be introduced into themolecule by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues, as described below.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, chloroacetyl phosphate, N-alkylmaleimides,3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide,p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect or reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing primary amine containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the argininealpha-amino group.

Tyrosyl residues are well-known targets of modification for introductionof spectral labels by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction carbodiimide (R'--N--C--N--R') such as1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residue are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful, for example, forcross-linking the component peptides of the complexes to each other orthe complex to a water-insoluble support matrix or to othermacromolecular carriers. Commonly used cross-linking agents include, forexample, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3- p-azidophenyl) dithiolpropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (Creighton, T. E., Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the Nterminal amine, and, in some instances, amidation ofthe C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility,absorption, biological half life, and the like. The moieties mayalternatively eliminate or attenuate any undesirable side effect of theprotein complex and the like. Moieties capable of mediating such effectsare disclosed, for example, in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990).

The term "fragment" is used to indicate a polypeptide derived from theamino acid sequence of the proteins, of the complexes having a lengthless than the full-length polypeptide from which it has been derived.Such a fragment may, for example, be produced by proteolytic cleavage ofthe full-length protein. Preferably, the fragment is obtainedrecombinantly by appropriately modifying the DNA sequence encoding theproteins to delete one or more amino acids at one or more sites of theC-terminus, N-terminus, and/or within the native sequence. Fragments ofa protein, when present in a complex resembling the naturally occurringcomplex, are useful for screening for compounds that act to modulatesignal transduction, as described below. It is understood that suchfragments, when present in a complex may retain one or morecharacterizing portions of the native complex. Examples of such retainedcharacteristics include: catalytic activity; substrate specificity;interaction with other molecules in the intact cell; regulatoryfunctions; or binding with an antibody specific for the native complex,or an epitope thereof.

Another functional derivative intended to be within the scope of thepresent invention is a complex comprising at least one "variant"polypeptide which either lack one or more amino acids or containadditional or substituted amino acids relative to the nativepolypeptide. The variant may be derived from a naturally occurringcomplex component by appropriately modifying the protein DNA codingsequence to add, remove, and/or to modify codons for one or more aminoacids at one or more sites of the C-terminus, N-terminus, and/or withinthe native sequence. It is understood that such variants having added,substituted and/or additional amino acids retain one or morecharacterizing portions of the native complex, as described above.

A functional derivative of complexes comprising proteins with deleted,inserted and/or substituted amino acid residues may be prepared usingstandard techniques well-known to those of ordinary skill in the art.For example, the modified components of the functional derivatives maybe produced using site-directed mutagenesis techniques (as exemplifiedby Adelman et al., 1983, DNA 2:183) wherein nucleotides in the DNAcoding the sequence are modified such that a modified coding sequence ismodified, and thereafter expressing this recombinant DNA in aprokaryotic or eukaryotic host cell, using techniques such as thosedescribed above. Alternatively, components of functional derivatives ofcomplexes with amino acid deletions, insertions and/or substitutions maybe conveniently prepared by direct chemical synthesis, using methodswell-known in the art. The functional derivatives of the complexestypically exhibit the same qualitative biological activity as the nativecomplexes.

XIX. Evaluation of Disorders

The protein complexes of the invention involved in disorders may beutilized in developing a prognostic evaluation of the condition of apatient suspected of exhibiting such a disorder. For example, biologicalsamples obtained from patients suspected of exhibiting a disorderinvolving a protein complex may be assayed for the presence of suchcomplexes. If such a protein complex is normally present, and thedevelopment of the disorder is caused by an abnormal quantity of thecomplex, the assay should compare complex levels in the biologicalsample to the range expected in normal tissue of the same cell type.

Among the assays which may be undertaken may include, but are notlimited to isolation of the protein complex of interest from thebiological sample, or assaying for the presence of the complex byexposing the sample to an antibody specific for the complex, butnon-reactive to any single, non-complexed component, and detectingwhether antibody has specifically bound.

Alternatively, one or more of the components of the protein complex maybe present in an abnormal level or in a modified form, relative to thelevel or form expected is normal, nononcogenic tissue of the same celltype. It is possible that overexpression of both components may indicatea particularly aggressive disorder. Thus, an assessment of theindividual and levels of mRNA and protein in diseased tissue cells mayprovide valuable clues as to the course of action to be undertaken intreatment of such a disorder. Assays of this type are well known tothose of skill in the art, and may include, but are not limited to,Northern blot analysis, RNAse protection assays, and PCR for determiningmRNA levels. Assays determining protein levels are also well known tothose of skill in the art, and may include, but are not limited to,Western blot analysis, immunoprecipitation, and ELISA analysis. Each ofthese techniques may also reveal potential differences in the form(e.g., the primary, secondary, or tertiary amino acid sequence, and/orpost-translational modifications of the sequence) of the component(s).

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the procedures used to identify thefull-length nucleic and amino acid sequences of PYK-2.

Materials and Methods

Chemicals

Bradykinin, pertusis toxin, cholera toxin, forskolin, phorbol12-myristate 13-acetate (PMA), calcium ionophore A23187, carbachol,muscarine, atrophine, mecamylamine, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were purchased from Sigma.

Cloning of PYK2 cDNA

RNA from rat spinal cord was used to prepare cDNA utilizing the reversetranscriptase of Molony murine leukemia virus (^(BRL)) according to themanufacturer's protocol. The cDNA was amplified by PCR utilizingdegenerate oligonucleotides primers corresponding to conserved tyrosinekinase motifs from subdomains TK6 and TK9 of PYK1; (the sense andantisense primers correspond to amino acid sequences IHRDLAARN SEQ ID NO3! and WMFGVTLW SEQ ID NO 4! respectively). The PCR was carried outunder the following conditions; 1 min at 94° C.; 1 min at 50° C. and 1min at 68° C. for 35 cycles. PCR products were electrophoresed, checkedby the size (˜210bp), purified and subcloned into pBluescript(Stratagene). Novel clones were screened by DNA sequencing. The cDNAinsert of clone #38 was used as probe to screen human brain cDNAlibraries (human fetal brain λgt 10 and human brain λgt 11, 6×10⁵recombinant clones each) essentially as described by Maniatis ().

DNA Sequencing and analysis

DNA sequencing was performed on both strands utilizing series ofoligonucleotide primers and subclones. The nucleotide sequence and thededuced amino acid sequence were subjected to homology search withGenbank and PIR databases using FASTA and BLAST mail-server program.

Northern blot analysis

Total RNA was isolated from mouse tissues by the acid guanidiniumthicynate-phenol-chloroform method (Anal. Biochem. 162; 156, 1987). Poly(A)⁺ RNA was denaturated with formaldehyde and electrophoresed on a 1%agarose/0.7% formaldehyde gel. RNAs were transferred to a nitrocellulosemembrane and hybridized with ³² p-labeled probe that contained the cDNAinsert of clone #38 as described above.

Antibodies

Antibodies against PYK2 were raised in rabbits immunized (HTl) either byGST fusion protein containing residues 362-647 or PYK2 or by syntheticpeptide corresponding to the 15 amino acids at the N-terminal end ofPYK2. Antisera were checked by immunoprecipitation and immunoblotanalysis, and the specificity was confirmed either by reactivity to therelated protein Fak or by competition with the antigenic or controlpeptides.

Cells and cell culture

PC12-rat pheochromocytoma cells were cultured in Dulbecco's modifiedEagle's medium containing 10% horse serum, 5% fetal bovine serum, 10μg/ml streptomycin and 100 units of penicillin/ml. NIH3T3, 293, GP+E-86and PA317 cells were grown in Dulbecco's modified Eagle's mediumcontaining 10% fetal bovine serum, 100 μg/ml streptomycin and 100 unitsof penicillin/ml.

Transfections and infections

For stable expression in PC12 cells, PYK2 was subcloned into theretroviral vector pLXSN (Miller and Rosman, Biotechniques 7:980, 1989).The construct was used to transfect GP+E-86 cells using lipofectiminereagent (GIBCO BRL). 48 hours after transfection, virus containingsupernatants were collected. Pure retrovirus-containing cell-freesupernatant were added to PC12 cells in the presence of polybrene (8μg/ml, Aldrich) for 4 hours (MCB 12 491, 1992). After 24 hours, infectedPC12 cells were split into growing medium containing 350 μl/mg G418.G418 resistant colonies were isolated two to three weeks later and thelevel of expression was determined by western blot analysis.

Stable cell lines of NIH3T3 that overexpress PYK2 were established bycontransfection of PYK2 subcloned into pLSV together with pSV2neoutilizing lipofectamine reagent (GIBCO BRL). Following transfection thecells were grown in Dulbecco's modified Eagle's medium containing 10%fetal bovine serum and 1 mg/ml G418. Transient transfections into 293cells were performed by using the calcium phosphate technique (*).

Constructs

GST-PYK2-- a DNA fragment of λ900 bp corresponding to residues 362-647of PYK2 was amplified by PCR utilizing the following oligonucleotideprimers: 5'-CGGGATCCTCATCATCCATCCTAGGAAAGA-3' (sense) SEQ ID NO 5! and5'-CGGGAATTCGTCGTAGTCCCAGCAGCGGGT-3' (antisense) SEQ ID NO 6!.

The PCR product was digested with HamHI and ECORI and subcloned intopGEX3X (Pharmacia). Expression of GST-PYK2 fusion protein was induced bythe 1 mM IPTG essentially as described by Smith et al.,(Gene 67:31,1988). The fusion protein was isolated by electroelution from SDS-PAGE.

PYK2-- The full length cDNA sequence of PYK2 was subcloned into thefollowing mammalian expression vectors: PLSV; downstream the SV40 earlypromoter, pLXSN-retroviral vector; downstream the Mo-MuLV long terminalrepeat; pRK5; downstream the CMV promoter.

PYK2-HA-- the influenza virus hemagglutinin peptide (YPYDVPDYAS) SEQ IDNO 7! was fused to the C-terminal end of PYK2 utilizing the followingoligonucleotide primers in the PCR: 5'-CACAATGTCTTCAAACGCCAC-3' SEQ IDNO 8! and 5'-GGCTCTAGATCACGATGCGTAGTCAGGGACATCGTATGGGRACTCTGCAGGTGGGTGGGCCAG-3'. SEQ ID NO 9! The amplified fragment was digested with RsrIIand Xbal and used to substitute the corresponding fragment of PYK2. Thenucleotide sequence of the final construct was confirmed by DNAsequencing.

Kinase negative mutant-- in order to construct a kinase negative mutant,Lys (457) was substituted to Ala by site directed mutagenesis utilizingthe `Transformer Site-Directed Mutagenesis Kit` (Clontech). Theoligonucleotide sequence was designed to create a new restriction siteof Nrul. The nucleotide sequence of the mutant was confirmed by DNAsequencing. The oligonucleotide sequence that was used for mutagenesisis: 5'-CAATGTAGCTGTCGCGACCTGCAAGAAAGAC-3' SEQ ID NO 10! (Nrulsite--bold, Lys-AAC substituted to Ala-GCG underline).

Rak-HA-- The rak cDNA subcloned in pBluescript was obtained fromBernardu Rudi (N.Y.U. medical center). The influenza virus hemagglutininpeptide was fused to the C-terminal end of Rak essentially as describedfor PYK2. The oligonucleotide primers that were used in the PCR were:5'-GCCAGCAGGCCATGTCACTGG-3' SEQ ID NO 11! and5'-CGGAATTCTTACGATGCGTAGTCAGGGACATCGTATGGGTAGACATCAGTTAACAT TTTG-3'. SEQID NO 12! The par product was digested with ball and EcoRI and was usedto substitute the corresponding fragment at the C-terminal end of Rak.The Rak-HA CDNA was subcloned into pRK5 downstream the CMV promotor andinto the retroviral vector pLXSN, downstream the Mo-MuLV long terminalrepeat.

Immunoprecipitation and Immunoblot Analysis

Cells were lysed in lysis buffer containing 50 mMN-2-hydroxyethylpiperazine-N'-2-ethanesulferic acid (HEPES pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mMethyleneglycol-bis (β-aminoethyl ether)-N,N,N'N'-tetraacetic acid(EGTA), 10 μg leupeptin per ml, 10 μg aprotinin per ml, 1 mMphenylmethylsulfonyl fluoride (PMSF), 200 μM sodium orthovanadate and100 mM sodium fluoride. Immunoprecipitations were performed usingprotein A-sepharose (Pharmacia) coupled to specific antibodies.Immunoprecipitates were washed either with HNTG' solution (20 mM HEPESbuffer at pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 100 mMsodium fluoride, 200 μM sodium orthovanadate) or successively with H'solution (50 mM Tris-HCl pH8, 500 mM NaCl, 0.1% SDS, 0.2% Triton X-100,100 mM NaF, 200 μM sodium orthovanadate) and L' solution (10 mM Tris-HClpH 8, 0.1% Triton X-100, 100 mM NaF, 200 μM sodium orthovanadate).

The washed immunoprecipitates were incubated for 5 min with gel samplebuffer at 100° C. and analyzed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). In some experiments the gel-embeddedproteins were electrophoretically transferred onto nitrocellulose. Theblot was then blocked with TBS (10 mM Tris pH 7.4, 150 mM NaCl) thatcontained 5%. low fat milk and 1% ovalbumin. Antisera or purified mAbswere then added in the same solution and incubation was carried out for1 h at 22° C. For detection the filters were washed three times (5 mineach wash) with TBS/0.05% Tween-20 and reacted for 45 min at roomtemperature with horseradish peroxidase-conjugated protein A. The enzymewas removed by washing as described above, and the filters were reactedfor 1 min with a chemiluminescence reagent (ECL, Amersham) and exposedto an autoradiography film for 1-15 min.

In vitro kinase assay

This was carried out on immunoprecipitates in 50 μl HNTG (20 mM Hepes pH7.5, 150 mM NaCl, 20% glycerol, 0.1% Triton X-100) containing 10 mMMnCl₂ and 5 μCi or mN-³² P!ATP for 20 min at 22° C. The samples werewashed with H', M' and, L' washing solutions, boiled for 5 min in samplebuffer and separated by SDS-PAGE.

Isolation of ACK/PYK

ACK/PYK may be isolated as described in Manser et al., Nature,363:364-367, 1993. Comparison analysis of the full length sequence ofACK/PYK with other tyrosine kinases indicates that is not closelyrelated to any of these, although it has some similarity to the focaladhesion kinase. Therefore, ACK/PYK represents a separate class oftyrosine kinases and isolation of related genes that belong to the sameclass is a major accomplishment.

Example 1

Isolation of PYK-2 cDNA

To identify genes related to the ACK/PYK protein tyrosine kinase, thepolymerase chain reaction (PCR) was applied in combination withdegenerated oligonucleotide primers based upon conserved motifs of thekinase domain of PTKs.

Oligonucleotides primers specifically designed to a highly conservedN-terminal motif of PTKs within subdomain TK6 (IHRDLAARN) SEQ ID NO 13.and ACK/PYK specific C-terminal primers within subdomain TK9 (WMFGVTLW)SEQ ID NO 14. were utilized. The amplification reactions with cDNAtemplates from 8 different sources gave rise to fragments of 0.2-0.9 kb.The PCR products were subcloned into pBlueScript and screened by DNAsequencing and hybridization under low stringency conditions.

A cDNA fragment of 210 bp from rat spinal cord was identified which ishighly related to the Focal Adhesion Kinase (FAK). The fragment wassequenced in the 3' and 5' directions and was subsequently used as aprobe to screen cDNA libraries (human fetal brain λgt 10 and human brainλgt 11, 6×10⁵ recombinant clones each).

Several overlapping clones spreading 1.5-3 kb were isolated and theircDNA inserts were analyzed by PCR, restriction mapping and sequencing.Two clones (#1 and #11) were chosen for further analysis and subcloning.Clone #1 contains an insert of 2.7 kb from the 5' end of the gene, andclone #11 contains an insert of 3 kb from the 3' end of the gene.

By utilizing a series of subclones and synthesized oligonucleotideprimers the full length sequence of PYK-2 was determined. The sequenceanalysis resulted in a composite sequence of 3309 bp long which containsa 104 bp 5' untranslated region, a 3021 bp coding region and 184 bp 3'untranslated region. The ATG encoding the translation initiation codonis preceded by four translation stop codons in all reading frames.

The long open reading frame encodes a protein of 1007 amino acids(predicted molecular mass of 110,770d) whose structural organization isvery similar to FAK. The PYK-2 protein contains a long N-terminalsequence of 422 amino acids followed by a tyrosine kinase catalyticdomain. The PYK-2 protein also contains the structural motifs common toall PTKS, two proline rich domains (19.6% and 17% proline respectively)and a focal adhesion targeting (FAT) motif in the C-terminal end.Comparison analysis of the amino acid sequence of PYK-2 with the humanFAK revealed 52% identity between the two proteins. The kinase domainand the FAT sequence are most closely related (62% homology).

The PYK-2 protein contains several predicted binding sites forintracellular substrates. For example, YLMV SEQ ID NO 15! is a predictedbinding site for GRB2 SH2 domain--tyrosine 879 of PYK-2. YVVV SEQ ID NO16! is a predicted binding site for SHPTP2 --tyrosine 903 of PYK-2.There are predicted phosphorylation sites for PKC, PKA and Ca/Calmodulinkinase. In addition, tyrosine 402 is a predicted autophosphorylationsite of PYK-2 and it may be involved in the binding of a src SH2 domain.This is based on the homology between tyrosine 397 of FAK which wasmapped as a major autophosphorylation site both in vivo and in vitro.This tyrosine provides an high affinity binding site for a src SH2domain. Both tyrosine 397 of FAK and tyrosine 402 of PYK-2 are locatedat the juncture of the N-terminal and the catalytic domain and arefollowed by sequence (Y)AEI which is very similar to the consensus ofthe high affinity src SH2 domain binding peptide YEEI.

Example 2

Antibodies to PYK-2

Antibodies against PYK-2 were raised in rabbits immunized either withGST fusion protein containing residues of PYK-2 or with syntheticpeptide corresponding to the 15 amino acids at the N-terminal end ofPYK-2. The antibodies are specific to PYK-2 and they do not cross reactwith FAK.

Example 3

Pattern of PYK-2 Expression

The tissue distribution of PYK-2 expression was determined by Northernblot analysis. Poly(A)⁺ RNAs were purified from mouse tissues (liver,lung, spleen, kidney, heart, brain, skin, uterus) and hybridized withtwo different probes corresponding to two different regions of the PYK-2gene. The results were identical in both cases. A 4.2 kb PYK-2transcript is relatively abundant in the brain but was also found inlower levels in the spleen and in the kidney.

The expression of PYK-2 in different cell lines was analyzed by IP/IButilizing anti-PYK-2 antibodies directed to the kinase domain asdescribed previously (GST-PYK-2). The expression pattern is summarizedin table 1. Some of the interesting observations are a mobility shift ofPYK-2 after differentiation of CHRF and L8057 (premegakaryocyte celllines) by TPA, high expression of Fak and PYK-2 in different cell lines;and in XC cells (rat sarcoma) PYK-2 is phosphorylated on tyrosine.

Example 4

Properties of PYK-2 protein

In order to analyze the biochemical properties of PYK-2 the full lengthcDNA was subcloned into the two mammalian expression vectors RK5 andpLSV. In parallel, an expression vector encoding the PYK-2 protein fusedto the influenza virus hemagglutinin peptide was constructed. Thisconstruct was used to identify the protein utilizing anti-HA antibodies.

Example 5

Transient transfection

pLSV-PYK-2-HA was transfected into cos cells. The protein was expressedat the predicted molecular mass (˜116 kD) as determined by IP and IBwith anti-HA antibodies. The protein is an active kinase as determinedby in vitro kinase assay utilizing (λ³² P) ATP or an in vitro kinaseassay utilizing cold ATP and immunoblotting with anti-phosphotyrosineantibodies.

Example 6

Stable cell lines

The PYK-2 cDNA cloned in pLSV was cotransfected with pSV2neo into PC12cells and NIH3T3 in order to establish stable cell lines. G418 resistantcolonies were screened by immunoprecipitating and immunoblotting.

NIH3T3 cell lines were established that overexpress the PYK-2 and thePYK-2-HA protein. In these cells PYK-2 undergoes tyrosinephosphorylation in response to PDGF, EGF and aFGF. The level ofphosphorylation is not so high. The stronger effect is achieved by TPAtreatment (6 μM) after 15 min incubation at 37° C. as determined by timecourse analysis.

Example 7

PC12 cells

The phosphorylation of PYK-2 on tyrosine residue in response todifferent stimuli was analyzed by immunoprecipitation of PYK-2 andimmunoblotting with anti-phosphotyrosine antibodies and vice versa.

The following treatment were used: Bradykinin, TPA, forskolin,forskolin+TPA, bradykinin+forskolin, NGF, Neuropeptide Y. Cholera toxin,Cholera toxin+TPA, Cholera toxin+bradykinin, pertusis toxin, pertusistoxin+TPA, bradykinin+pertusis toxin, calcium ionophore A23187,bombesin.

The following results were obtained: PYK-2 undergoes tyrosinephosphorylation in response to TPA (1.6 μM 15 min at 37° C.), bradykinin(1 μM 1 min at 37° C.) and calcium ionophore A23187 (2 μM 15 min at 37°C.). Forskolin increase the response of TPA but does not give any signalby itself. Cholera toxin gave higher signal in combination with TPA andbradykinin but didn't cause phosphorylation of PYK-2 alone. Pertusistoxin also induced the response of TPA and bradykinin but didn't causeany response alone. In order to determined if the bradykinin effect ismediated by PKC signaling pathway, attempts to down regulate PKC bychronic treatment with TPA (twice) did not give a clear answer.

One interpretation of these results is that PKC and PKA (and maybeCa/calmudolin kinase) induce the autophosphorylation of PYK-2 inresponse to ser/the phosphorylation. This interpretation may be checkedby utilizing specific inhibitors to PKC and PKA and by phosphamino-acidanalysis.

Example 8

Phosphorylation of RAK

293 cells in 65 mm plates were transiently transfected either with thepotassium channel-RAK-HA alone, or together with Fak, PYK2 or thePYK2-kinase negative mutant (PKN). 12 hr following transfection thecells were grown in DMEM containing 0.3% fetal bovine serum for 24hours. The cells were either stimulated with PMA (1.6 μM) or withcalcium ionophore A23187 (6 μM) for 15 min at 37° C. or leftunstimulated. The cells were solubilized and the expression level ofeach protein was determined by western blot analysis. The Rak proteinwas immunoprecipitated by anti-HA antibodies and its phosphorylation ontyrosine residues was analyzed by western blot analysis utilizinganti-phosphotyrosine antibodies following immunoprecipitation of theproteins either with anti-PYK2 antibodies (for PYK2 and PKN) or withanti Fax antibodies for (Fak).

The expression level of each protein (Rak PYK2, PKN and Fak) and thetyrosine phosphorylation of Rak, PYK2, PKN and Fak were measured.

Only the kinase active PYK2 protein phosphorylated the potassiumchannel. No phosphorylation was observed with kinase negative PYK2 orwith FAK.

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1009                                                              (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      MetSerGlyValSerGluProLeuSerArgValLysLeuGlyThrLeu                              151015                                                                        ArgArgProGluGlyProAlaGluProMetValValValProValAsp                              202530                                                                        ValGluLysGluAspValArgIleLeuLysValCysPheTyrSerAsn                              354045                                                                        SerPheAsnProGlyLysAsnPheLysLeuValLysCysThrValGln                              505560                                                                        ThrGluIleArgGluIleIleThrSerIleLeuLeuSerGlyArgIle                              65707580                                                                      GlyProAsnIleArgLeuAlaGluCysTyrGlyLeuArgLeuLysHis                              859095                                                                        MetLysSerAspGluIleHisTrpLeuHisProGlnMetThrValGly                              100105110                                                                     GluValGlnAspLysTyrGluCysLeuHisValGluAlaGluTrpArg                              115120125                                                                     TyrAspLeuGlnIleArgTyrLeuProGluAspPheMetGluSerLeu                              130135140                                                                     LysGluAspArgThrThrLeuLeuTyrPheTyrGlnGlnLeuArgAsn                              145150155160                                                                  AspTyrMetGlnArgTyrAlaSerLysValSerGluGlyMetAlaLeu                              165170175                                                                     GlnLeuGlyCysLeuGluLeuArgArgPhePheLysAspMetProHis                              180185190                                                                     AsnAlaLeuAspLysLysSerAsnPheGluLeuLeuGluLysGluVal                              195200205                                                                     GlyLeuAspLeuPhePheProLysGlnMetGlnGluAsnLeuLysPro                              210215220                                                                     LysGlnPheArgLysMetIleGlnGlnThrPheGlnGlnTyrAlaSer                              225230235240                                                                  LeuArgGluGluGluCysValMetLysPhePheAsnThrLeuAlaGly                              245250255                                                                     PheAlaAsnIleAspGlnGluThrTyrArgCysGluLeuIleGlnGly                              260265270                                                                     TrpAsnIleThrValAspLeuValIleGlyProLysGlyIleArgGln                              275280285                                                                     LeuThrSerGlnAspAlaLysProThrCysLeuAlaGluPheLysGln                              290295300                                                                     IleArgSerIleArgCysLeuProLeuGluGluGlyGlnAlaValLeu                              305310315320                                                                  GlnLeuGlyIleGluGlyAlaProGlnAlaLeuSerIleLysThrSer                              325330335                                                                     SerLeuAlaGluAlaGluAsnMetAlaAspLeuIleAspGlyTyrCys                              340345350                                                                     ArgLeuGlnGlyGluHisGlnGlySerLeuIleIleHisProArgLys                              355360365                                                                     AspGlyGluLysArgAsnSerLeuProGlnIleProMetLeuAsnLeu                              370375380                                                                     GluAlaArgArgSerHisLeuSerGluSerCysSerIleGluSerAsp                              385390395400                                                                  IleTyrAlaGluIleProAspGluThrLeuArgArgProGlyGlyPro                              405410415                                                                     GlnTyrGlyIleAlaArgGluAspValValLeuAsnArgIleLeuGly                              420425430                                                                     GluGlyPhePheGlyGluValTyrGluGlyValTyrThrAsnHisLys                              435440445                                                                     GlyGluLysIleAsnValAlaValLysThrCysLysLysAspCysThr                              450455460                                                                     LeuAspAsnLysGluLysPheMetSerGluAlaValIleMetLysAsn                              465470475                                                                     LeuAspHisProHisIleValLysLeuIleGlyIleIleGluGluGlu                              480485490495                                                                  ProThrTrpIleIleMetGluLeuTyrProTyrGlyGluLeuGlyHis                              500505510                                                                     TyrLeuGluArgAsnLysAsnSerLeuLysValLeuThrLeuValLeu                              515520525                                                                     TyrSerLeuGlnIleCysLysAlaMetAlaTyrLeuGluSerIleAsn                              530535540                                                                     CysValHisArgAspIleAlaValArgAsnIleLeuValAlaSerPro                              545550555                                                                     GluCysValLysLeuGlyAspPheGlyLeuSerArgTyrIleGluAsp                              560565570575                                                                  GluAspTyrTyrLysAlaSerValThrArgLeuProIleLysTrpMet                              580585590                                                                     SerProGluSerIleAsnPheArgArgPheThrThrAlaSerAspVal                              595600605                                                                     TrpMetPheAlaValCysMetTrpGluIleLeuSerPheGlyLysGln                              610615620                                                                     ProPhePheTrpLeuGluAsnLysAspValIleGlyValLeuGluLys                              625630635                                                                     GlyAspArgLeuProLysProAspLeuCysProProValLeuTyrThr                              640645650                                                                     LeuMetThrArgCysTrpAspTyrAspProSerAspArgProArgPhe                              655660665670                                                                  ThrGluLeuValCysSerLeuSerAspValTyrGlnMetGluLysAsp                              675680685                                                                     IleAlaMetGluGlnGluArgAsnAlaArgTyrArgThrProLysIle                              690695700                                                                     LeuGluProThrAlaPheGlnGluProProProLysProSerArgPro                              705710715                                                                     LysTyrArgProProProGlnThrAsnLeuLeuAlaProLysLeuGln                              720725730                                                                     PheGlnValProGluGlyLeuCysAlaSerSerProThrLeuThrSer                              735740745750                                                                  ProMetGluTyrProSerProValAsnSerLeuHisThrProProLeu                              755760765                                                                     HisArgHisAsnValPheLysArgHisSerMetArgGluGluAspPhe                              770775780                                                                     IleGlnProSerSerArgGluGluAlaGlnGlnLeuTrpGluAlaGlu                              785790795                                                                     LysValLysMetArgGlnIleLeuAspLysGlnGlnLysGlnMetVal                              800805810                                                                     GluAspTyrGlnTrpLeuArgGlnGluGluLysSerLeuAspProMet                              815820825830                                                                  ValTyrMetAsnAspLysSerProLeuThrProGluLysGluValGly                              835840845                                                                     TyrLeuGluPheThrGlyProProGlnLysProProArgLeuGlyAla                              850855860                                                                     GlnSerIleGlnProThrAlaAsnLeuAspArgThrAspAspLeuVal                              865870875                                                                     TyrLeuAsnValMetGluLeuValArgAlaValLeuGluLeuLysAsn                              880885890                                                                     GluLeuCysGlnLeuProProGluGlyTyrValValValValLysAsn                              895900905910                                                                  ValGlyLeuThrLeuArgLysLeuIleGlySerValAspAspLeuLeu                              915920925                                                                     ProSerLeuProSerSerSerArgThrGluIleGluGlyThrGlnLys                              930935940                                                                     LeuLeuAsnLysAspLeuAlaGluLeuIleAsnLysMetArgLeuAla                              945950955                                                                     GlnGlnAsnAlaValThrSerLeuSerGluGluCysLysArgGlnMet                              960965970                                                                     LeuThrAlaSerHisThrLeuAlaValAspAlaLysAsnLeuLeuAsp                              975980985990                                                                  AlaValAspGlnAlaLysValLeuAlaAsnLeuAlaHisProProAla                              99510001005                                                                   Glu                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3416                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: nucleic                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CGGTACAGGTAAGTCGGCCGGGCAGGTAGGGGTGCCCGAGGAGTAGTCGCTGGAGTCCGC60                GCCTCCCTGGGACTGCAATGTGCCGGTCTTAGCTGCTGCCTGAGAGGATGTCTGGGGTGT120               CCGAGCCCCTGAGCCGAGTAAAGTTGGGCACATTACGCCGGCCTGAAGGCCCTGCAGAGC180               CCATGGTGGTGGTACCAGTAGATGTGGAAAAGGAGGACGTGCGTATCCTCAAGGTCTGCT240               TCTATAGCAACAGCTTCAATCCTGGGAAGAACTTCAAACTGGTCAAATGCACTGTCCAGA300               CGGAGATCCGGGAGATCATCACCTCCATCCTGCTGAGCGGGCGGATCGGGCCCAACATCC360               GGTTGGCTGAGTGCTATGGGCTGAGGCTGAAGCACATGAAGTCCGATGAGATCCACTGGC420               TGCACCCACAGATGACGGTGGGTGAGGTGCAGGACAAGTATGAGTGTCTGCACGTGGAAG480               CCGAGTGGAGGTATGACCTTCAAATCCGCTACTTGCCAGAAGACTTCATGGAGAGCCTGA540               AGGAGGACAGGACCACGCTGCTCTATTTTTACCAACAGCTCCGGAACGACTACATGCAGC600               GCTACGCCAGCAAGGTCAGCGAGGGCATGGCCCTGCAGCTGGGCTGCCTGGAGCTCAGGC660               GGTTCTTCAAGGATATGCCCCACAATGCACTTGACAAGAAGTCCAACTTCGAGCTCCTAG720               AAAAGGAAGTGGGGCTGGACTTGTTTTTCCCAAAGCAGATGCAGGAGAACTTAAAGCCCA780               AACAGTTCCGGAAGATGATCCAGCAGACCTTCCAGCAGTACGCCTCGCTCAGGGAGGAGG840               AGTGCGTCATGAAGTTCTTCAACACTCTCGCCGGCTTCGCCAACATCGACCAGGAGACCT900               ACCGCTGTGAACTCATTCAAGGATGGAACATTACTGTGGACCTGGTCATTGGCCCTAAAG960               GGATCCGCCAGCTGACTAGTCAGGACGCAAAGCCCACCTGCCTGGCCGAGTTCAAGCAGA1020              TCAGGTCCATCAGGTGCCTCCCGCTGGAGGAGGGCCAGGCAGTACTTCAGCTGGGCATTG1080              AAGGTGCCCCCCAGGCCTTGTCCATCAAAACCTCATCCCTAGCAGAGGCTGAGAACATGG1140              CTGACCTCATAGACGGCTACTGCCGGCTGCAGGGTGAGCACCAAGGCTCTCTCATCATCC1200              ATCCTAGGAAAGATGGTGAGAAGCGGAACAGCCTGCCCCAGATCCCCATGCTAAACCTGG1260              AGGCCCGGCGGTCCCACCTCTCAGAGAGCTGCAGCATAGAGTCAGACATCTACGCAGAGA1320              TTCCCGACGAAACCCTGCGAAGGCCCGGAGGTCCACAGTATGGCATTGCCCGTGAAGATG1380              TGGTCCTGAATCGTATTCTTGGGGAAGGCTTTTTTGGGGAGGTCTATGAAGGTGTCTACA1440              CAAATCACAAAGGGGAGAAAATCAATGTAGCTGTCAAGACCTGCAAGAAAGACTGCACTC1500              TGGACAACAAGGAGAAGTTCATGAGCGAGGCAGTGATCATGAAGAACCTCGACCACCCGC1560              ACATCGTGAAGCTGATCGGCATCATTGAAGAGGAGCCCACCTGGATCATCATGGAATTGT1620              ATCCCTATGGGGAGCTGGGCCACTACCTGGAGCGGAACAAGAACTCCCTGAAGGTGCTCA1680              CCCTCGTGCTGTACTCACTGCAGATATGCAAAGCCATGGCCTACCTGGAGAGCATCAACT1740              GCGTGCACAGGGACATTGCTGTCCGGAACATCCTGGTGGCCTCCCCTGAGTGTGTGAAGC1800              TGGGGGACTTTGGTCTTTCCCGGTACATTGAGGACGAGGACTATTACAAAGCCTCTGTGA1860              CTCGTCTCCCCATCAAATGGATGTCCCCAGAGTCCATTAACTTCCGACGCTTCACGACAG1920              CCAGTGACGTCTGGATGTTCGCCGTGTGCATGTGGGAGATCCTGAGCTTTGGGAAGCAGC1980              CCTTCTTCTGGCTGGAGAACAAGGATGTCATCGGGGTGCTGGAGAAAGGAGACCGGCTGC2040              CCAAGCCTGATCTCTGTCCACCGGTCCTTTATACCCTCATGACCCGCTGCTGGGACTACG2100              ACCCCAGTGACCGGCCCCGCTTCACCGAGCTGGTGTGCAGCCTCAGTGACGTTTATCAGA2160              TGGAGAAGGACATTGCCATGGAGCAAGAGAGGAATGCTCGCTACCGAACCCCCAAAATCT2220              TGGAGCCCACAGCCTTCCAGGAACCCCCACCCAAGCCCAGCCGACCTAAGTACAGACCCC2280              CTCCGCAAACCAACCTCCTGGCTCCAAAGCTGCAGTTCCAGGTTCCTGAGGGTCTGTGTG2340              CCAGCTCTCCTACGCTCACCAGCCCTATGGAGTATCCATCTCCCGTTAACTCACTGCACA2400              CCCCACCTCTCCACCGGCACAATGTCTTCAAACGCCACAGCATGCGGGAGGAGGACTTCA2460              TCCAACCCAGCAGCCGAGAAGAGGCCCAGCAGCTGTGGGAGGCTGAAAAGGTCAAAATGC2520              GGCAAATCCTGGACAAACAGCAGAAGCAGATGGTGGAGGACTACCAGTGGCTCAGGCAGG2580              AGGAGAAGTCCCTGGACCCCATGGTTTATATGAATGATAAGTCCCCATTGACGCCAGAGA2640              AGGAGGTCGGCTACCTGGAGTTCACAGGGCCCCCACAGAAGCCCCCGAGGCTGGGCGCAC2700              AGTCCATCCAGCCCACAGCTAACCTGGACCGGACCGATGACCTGGTGTACCTCAATGTCA2760              TGGAGCTGGTGCGGGCCGTGCTGGAGCTCAAGAATGAGCTCTGTCAGCTGCCCCCCGAGG2820              GCTACGTGGTGGTGGTGAAGAATGTGGGGCTGACCCTGCGGAAGCTCATCGGGAGCGTGG2880              ATGATCTCCTGCCTTCCTTGCCGTCATCTTCACGGACAGAGATCGAGGGCACCCAGAAAC2940              TGCTCAACAAAGACCTGGCAGAGCTCATCAACAAGATGCGGCTGGCGCAGCAGAACGCCG3000              TGACCTCCCTGAGTGAGGAGTGCAAGAGGCAGATGCTGACGGCTTCACACACCCTGGCTG3060              TGGACGCCAAGAACCTGCTCGACGCTGTGGACCAGGCCAAGGTTCTGGCCAATCTGGCCC3120              ACCCACCTGCAGAGTGACGGAGGGTGGGGGCCACCTGCCTGCGTCTTCCGCCCCTGCCTG3180              CCATGTACCTCCCCTGCCTTGCTGTTGGTCATGTGGGTCTTCCAGGGAGAAGGCCAAGGG3240              GAGTCACCTTCCCTTGCCACTTTGCACGACGCCCTCTCCCCACCCCTACCCCTGGCTGTA3300              CTGCTCAGGCTGCAGCTGGACAGAGGGGACTCTGGGCTATGGACACAGGGTGACGGTGAC3360              AAAGATGGCTCAGAGGGGGACTGCTGCTGCCTGGCCACTGCTCCCTAAGCCAGCCT3416                  (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      IleHisArgAspLeuAlaAlaArgAsn                                                   (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      TrpMetPheGlyValThrLeuTrp                                                      5                                                                             (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      CGGGATCCTCATCATCCATCCTAGGAAAGA30                                              (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      CGGGAATTCGTCGTAGTCCCAGCAGCGGGT30                                              (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      TyrProTyrAspValProAspTyrAlaSer                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      CACAATGTCTTCAAACGCCAC21                                                       (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GGCTCTAGATCACGATGCGTAGTCAGGGACATCGTATGGGRACTCTGCAGGTGGGTGGGC60                CAG63                                                                         (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     CAATGTAGCTGTCGCGACCTGCAAGAAAGAC31                                             (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GCCAGCAGGCCATGTCACTGG21                                                       (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     CGGAATTCTTACGATGCGTAGTCAGGGACATCGTATGGGTAGACATCAGTTAACATTTTG60                (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     IleHisArgAspLeuAlaAlaArgAsn                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     TrpMetPheGlyValThrLeuTrp                                                      5                                                                             (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     TyrLeuMetVal                                                                  (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     TyrValValVal                                                                  __________________________________________________________________________

What is claimed is:
 1. An isolated or purified nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is isolated.
 3. An isolated or purified nucleic acid molecule comprising at least one hundred and five nucleotides of said molecule of claim
 1. 4. The nucleic acid molecule of claim 3, wherein said nucleic acid molecule is present in a sample at least from 10% to 20% more frequently than any other nucleotide sequence.
 5. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is purified.
 6. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule is at least two times more pure on a mg/ml basis than in its natural environment.
 7. The nucleic acid molecule of claim 6, wherein said nucleic acid molecule is at least five times more pure on a mg/ml basis than in its natural environment.
 8. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
 9. The nucleic acid molecule of claim 1, said nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2.
 10. A recombinantly made nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1, said nucleic acid molecule operably linked to a regulatory sequence to allow expression of said nucleic acid molecule.
 11. The nucleic acid molecule of claim 10, wherein said nucleic acid molecule encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
 12. The nucleic acid molecule of claim 10, said nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2.
 13. The nucleic acid molecule of claim 12, said nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:2.
 14. A genetically engineered host cell containing a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1, said nucleotide sequence operably linked to a regulatory sequence to allow expression of said nucleotide sequence.
 15. The host cell of claim 14, wherein said nucleic acid molecule encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
 16. The host cell of claim 14, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2.
 17. The host cell of claim 16, wherein said nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO:2.
 18. The host cell of claim 14, wherein said host cell is prokaryotic.
 19. The host cell of claim 14, wherein said host cell is eukaryotic.
 20. An isolated, purified, or recombinantly made nucleic acid molecule comprising at least one hundred and five nucleotides of the nucleic acid sequence of SEQ ID NO:2, said nucleic acid molecule encoding a polypeptide possessing at least one of the functional activities of the amino acid sequence of SEQ ID NO:1, wherein said actives are phosphorylation and regulation of potassium channels.
 21. An expression construct containing the nucleic acid molecule of claim
 20. 22. A genetically engineered host cell containing the nucleotide sequence of claim 20, said nucleotide sequence operably linked to a regulatory sequence to allow expression of said nucleotide sequence.
 23. The host cell of claim 22, wherein said host cell is prokaryotic.
 24. The host cell of claim 22, wherein said host cell is eukaryotic.
 25. The nucleic acid molecule of claim 9, said nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:2. 